Zebularine reactivates silenced E-cadherin but unlike 5-Azacytidine does not induce switching from latent to lytic Epstein-Barr virus infection in Burkitt's lymphoma Akata cells

Epigenetic silencing of regulatory genes by aberrant methylation contributes to tumorigenesis. DNA methyltransferase inhibitors (DNMTI) represent promising new drugs for anti-cancer therapies. The DNMTI 5-Azacytidine is effective against myelodysplastic syndrome, but induces switching of latent to lytic Epstein-Barr virus (EBV) in vitro and results in EBV DNA demethylation with the potential of induction of lytic EBV in vivo. This is of considerable concern given that recurrent lytic EBV has been linked with an increased incidence of EBV-associated lymphomas. Based on the distinct properties of action we hypothesized that the newer DNMTI Zebularine might differ from 5-Azacytidine in its potential to induce switching from latent to lytic EBV. Here we show that both 5-Azacytidine and Zebularine are able to induce expression of E-cadherin, a cellular gene frequently silenced by hypermethylation in cancers, and thus demonstrate that both DNMTI are active in our experimental setting consisting of EBV-harboring Burkitt's lymphoma Akata cells. Quantification of mRNA expression of EBV genes revealed that 5-Azacytidine induces switching from latent to lytic EBV and, in addition, that the immediate-early lytic infection progresses to early and late lytic infection. Furthermore, 5-Azacytidine induced upregulation of the latent EBV genes LMP2A, LMP2B, and EBNA2 in a similar fashion as observed following switching of latent to lytic EBV upon cross-linking of the B-cell receptor. In striking contrast, Zebularine did not exhibit any effect neither on lytic nor on latent EBV gene expression. Thus, Zebularine might be safer than 5-Azacytidine for the treatment of cancers in EBV carriers and could also be applied against EBV-harboring tumors, since it does not induce switching from latent to lytic EBV which may result in secondary EBV-associated malignancies

Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray

BACKGROUND: The Epstein-Barr virus (EBV) is associated with lymphoid malignancies, including Burkitt's lymphoma (BL), and can transform human B cells in vitro. EBV-harboring cell lines are widely used to investigate lymphocyte transformation and oncogenesis. Qualitative EBV gene expression has been extensively described, but knowledge of quantitative transcription is lacking. We hypothesized that transcription levels of EBNA1, the gene essential for EBV persistence within an infected cell, are similar in BL cell lines. RESULTS: To compare quantitative gene transcription in the BL cell lines Namalwa, Raji, Akata, Jijoye, and P3HR1, we developed an oligonucleotide microarray chip, including 17 housekeeping genes, six latent EBV genes (EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1, LMP2), and four lytic EBV genes (BZLF1, BXLF2, BKRF2, BZLF2), and used the cell line B95.8 as a reference for EBV gene transcription. Quantitative polymerase chain reaction assays were used to validate microarray results. We found that transcription levels of housekeeping genes differed considerably among BL cell lines. Using a selection of housekeeping genes with similar quantitative transcription in the tested cell lines to normalize EBV gene transcription data, we showed that transcription levels of EBNA1 were quite similar in very different BL cell lines, in contrast to transcription levels of other EBV genes. As demonstrated with Akata cells, the chip allowed us to accurately measure EBV gene transcription changes triggered by treatment interventions. CONCLUSION: Our results suggest uniform EBNA1 transcription levels in BL and that microarray profiling can reveal novel insights on quantitative EBV gene transcription and its impact on lymphocyte biology

Laboratory-scale simulation and real-time tracking of a microbial contamination event and subsequent shock-chlorination in drinking water

Rapid contamination of drinking water in distribution and storage systems can occur due to pressure drop, backflow, cross-connections, accidents, and bio-terrorism. Small volumes of a concentrated contaminant (e.g., wastewater) can contaminate large volumes of water in a very short time with potentially severe negative health impacts. The technical limitations of conventional, cultivation-based microbial detection methods neither allow for timely detection of such contaminations, nor for the real-time monitoring of subsequent emergency remediation measures (e.g., shock-chlorination). Here we applied a newly developed continuous, ultra high-frequency flow cytometry approach to track a rapid pollution event and subsequent disinfection of drinking water in an 80-min laboratory scale simulation. We quantified total (TCC) and intact (ICC) cell concentrations as well as flow cytometric fingerprints in parallel in real-time with two different staining methods. The ingress of wastewater was detectable almost immediately (i.e., after 0.6% volume change), significantly changing TCC, ICC, and the flow cytometric fingerprint. Shock chlorination was rapid and detected in real time, causing membrane damage in the vast majority of bacteria (i.e., drop of ICC from more than 380 cells mu l(-1) to less than 30 cells mu l(-1) within 4 min). Both of these effects as well as the final wash-in of fresh tap water followed calculated predictions well. Detailed and highly quantitative tracking of microbial dynamics at very short time scales and for different characteristics (e.g., concentration, membrane integrity) is feasible. This opens up multiple possibilities for targeted investigation of a myriad of bacterial short-term dynamics (e.g., disinfection, growth, detachment, operational changes) both in laboratory-scale research and full-scale system investigations in practice

Ensuring microbial water quality for on-site water reuse: Importance of online sensors for reliable operation

A growing number of cities and regions are promoting or mandating on-site treatment and reuse of wastewater, which has resulted in the implementation of several thousand on-site water reuse systems on a global scale. However, there is only limited information on the (microbial) water quality from implemented systems. The focus of this study was on two best-in-class on-site water reuse systems in Bengaluru, India, which typically met the local water quality requirements during monthly compliance testing. This study aimed to (i) assess the microbial quality of the reclaimed water at a high temporal resolution (daily or every 15 min), and (ii) explore whether measurements from commercially available sensors can be used to improve the operation of such systems. The monitoring campaign revealed high variations in microbial water quality, even in these best-in-class systems, rendering the water inadequate for the intended reuse applications (toilet flushing and landscape irrigation). These variations were attributed to two key factors: (1) the low frequency of chlorination, and (2) fluctuations of the chlorine demand of the water, in particular of ammonium concentrations. Such fluctuations are likely inherent to on-site systems, which rely on a low level of process control. The monitoring campaign showed that the microbial water quality was most closely related to oxidation–reduction potential (ORP) and free chlorine sensors. Due to its relatively low cost and low need for maintenance, the ORP emerges as a compelling candidate for automating the chlorination to effectively manage variations in chlorine demand and ensure safe water reuse. Overall, this study underscores the necessity of integrating treatment trains, operation, and monitoring for safe on-site water reuse

Ensuring microbial water quality for on-site water reuse: Importance of online sensors for reliable operation

A growing number of cities and regions are promoting or mandating on-site treatment and reuse of wastewater, which has resulted in the implementation of several thousand on-site water reuse systems on a global scale. However, there is only limited information on the (microbial) water quality from implemented systems. The focus of this study was on two best-in-class on-site water reuse systems in Bengaluru, India, which typically met the local water quality requirements during monthly compliance testing. This study aimed to (i) assess the microbial quality of the reclaimed water at a high temporal resolution (daily or every 15 min), and (ii) explore whether measurements from commercially available sensors can be used to improve the operation of such systems. The monitoring campaign revealed high variations in microbial water quality, even in these best-in-class systems, rendering the water inadequate for the intended reuse applications (toilet flushing and landscape irrigation). These variations were attributed to two key factors: (1) the low frequency of chlorination, and (2) fluctuations of the chlorine demand of the water, in particular of ammonium concentrations. Such fluctuations are likely inherent to on-site systems, which rely on a low level of process control. The monitoring campaign showed that the microbial water quality was most closely related to oxidation–reduction potential (ORP) and free chlorine sensors. Due to its relatively low cost and low need for maintenance, the ORP emerges as a compelling candidate for automating the chlorination to effectively manage variations in chlorine demand and ensure safe water reuse. Overall, this study underscores the necessity of integrating treatment trains, operation, and monitoring for safe on-site water reuse.ISSN:2589-914

Evaluating Monitoring Strategies to Detect Precipitation-Induced Microbial Contamination Events in Karstic Springs Used for Drinking Water

Monitoring of microbial drinking water quality is a key component for ensuring safety and understanding risk, but conventional monitoring strategies are typically based on low sampling frequencies (e.g., quarterly or monthly). This is of concern because many drinking water sources, such as karstic springs are often subject to changes in bacterial concentrations on much shorter time scales (e.g., hours to days), for example after precipitation events. Microbial contamination events are crucial from a risk assessment perspective and should therefore be targeted by monitoring strategies to establish both the frequency of their occurrence and the magnitude of bacterial peak concentrations. In this study we used monitoring data from two specific karstic springs. We assessed the performance of conventional monitoring based on historical records and tested a number of alternative strategies based on a high-resolution data set of bacterial concentrations in spring water collected with online flow cytometry (FCM). We quantified the effect of increasing sampling frequency and found that for the specific case studied, at least bi-weekly sampling would be needed to detect precipitation events with a probability of >90%. We then proposed an optimized monitoring strategy with three targeted samples per event, triggered by precipitation measurements. This approach is more effective and efficient than simply increasing overall sampling frequency. It would enable the water utility to (1) analyze any relevant event and (2) limit median underestimation of peak concentrations to approximately 10%. We conclude with a generalized perspective on sampling optimization and argue that the assessment of short-term dynamics causing microbial peak loads initially requires increased sampling/analysis efforts, but can be optimized subsequently to account for limited resources. This offers water utilities and public health authorities systematic ways to evaluate and optimize their current monitoring strategies

Ectopic expression of human topoisomerase IIα fragments and etoposide resistance in mammalian cells

Cellular resistance to etoposide has been correlated both with reduced levels and an aberrant cytoplasmic accumulation of the drug`s target, topoisomerase IIα (topo IIα). It is not known, however, whether a cytoplasmic pool of topo IIα is sufficient to confer drug resistance on cultured mammalian cells. In our study, we have transfected mouse fibroblasts and human 293 cells with truncated forms of human topo IIα fused to GFP and have examined the transformants for the subcellular localization of topo IIα and their resistance to etoposide. Transient transfection resulted in high‐level expression of all GFP‐topo IIα fusions tested, whereas in stably transfected cells the levels varied significantly. Transfectants expressing a central or a carboxy‐terminal topo IIα domain (aa 428‐1504, 639‐1028 or 1028‐1504) accumulated high levels of the fusion proteins, while only very low amounts of GFP‐topo IIα proteins were observed in cell lines expressing constructs that retain the amino‐terminus of the enzyme (aa 1‐1214, aa 1‐939, aa 1‐611). Our results suggest that the topo IIα amino‐terminus affects the stability of truncated forms of the enzyme in mammalian cells, perhaps due to targeted degradation. Assays that screen for cell vitality and DNA synthesis reveal no significant changes in etoposide sensitivity in transfected cells expressing high levels of cytoplasmic or nuclear localized topo II fusion proteins. Retroviral expression of a cytoplasmically anchored domain of human topo IIα also failed to confer drug resistance. These results suggest that a cytoplasmic pool of topo IIα is not sufficient to render cultured mammalian cells drug resistant

The feasibility of automated online flow cytometry for in-situ monitoring of microbial dynamics in aquatic ecosystems

Fluorescent staining coupled with flow cytometry (FCM) is often used for the monitoring, quantification and characterization of bacteria in engineered and environmental aquatic ecosystems including seawater, freshwater, drinking water, wastewater, and industrial bioreactors. However, infrequent grab sampling hampers accurate characterization and subsequent understanding of microbial dynamics in all of these ecosystems. A logic technological progression is high throughput and full automation of the sampling, staining, measurement, and data analysis steps. Here we assess the feasibility and applicability of automated FCM by means of actual data sets produced with prototype instrumentation. As proof-of-concept we demonstrate examples of microbial dynamics in (i) flowing tap water from a municipal drinking water supply network and (ii) river water from a small creek subject to two rainfall events. In both cases, automated measurements were done at 15-min intervals during 12–14 consecutive days, yielding more than 1000 individual data points for each ecosystem. The extensive data sets derived from the automated measurements allowed for the establishment of baseline data for each ecosystem, as well as for the recognition of daily variations and specific events that would most likely be missed (or miss-characterized) by infrequent sampling. In addition, the online FCM data from the river water was combined and correlated with online measurements of abiotic parameters, showing considerable potential for a better understanding of cause-and-effect relationships in aquatic ecosystems. Although several challenges remain, the successful operation of an automated online FCM system and the basic interpretation of the resulting data sets represent a breakthrough toward the eventual establishment of fully automated online microbiological monitoring technologies

Latent Membrane Protein 2B Regulates Susceptibility to Induction of Lytic Epstein-Barr Virus Infection▿

The B-lymphotropic Epstein-Barr virus (EBV) encodes two isoforms of latent membrane protein 2 (LMP2), LMP2A and LMP2B, which are expressed during latency in B cells. The function of LMP2B is largely unknown, whereas LMP2A blocks B-cell receptor (BCR) signaling transduction and induction of lytic EBV infection, thereby promoting B-cell survival. Transfection experiments on LMP2B in EBV-negative B cells and the silencing of LMP2B in EBV-harboring Burkitt's lymphoma-derived Akata cells suggest that LMP2B interferes with the function of LMP2A, but the role of LMP2B in the presence of functional EBV has not been established. Here, LMP2B, LMP2A, or both were overexpressed in EBV-harboring Akata cells to study the function of LMP2B. The overexpression of LMP2B increased the magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking, as indicated by a more-enhanced upregulation and expression of EBV lytic genes and significantly increased production of transforming EBV compared to Akata vector control cells or LMP2A-overexpressing cells. Moreover, LMP2B lowered the degree of BCR cross-linking required to induce lytic EBV infection. Finally, LMP2B colocalized with LMP2A as demonstrated by immunoprecipitation and immunofluorescence and restored calcium mobilization upon BCR cross-linking, a signaling process inhibited by LMP2A. Thus, our findings suggest that LMP2B negatively regulates the function of LMP2A in preventing the switch from latent to lytic EBV replication