Epstein-Barr virus (EBV) is a human herpesvirus associated with B-cell and epithelial cell malignancies. EBV lytically infects normal differentiated oral epithelial cells, where it causes a tongue ...lesion known as oral hairy leukoplakia (OHL) in immunosuppressed patients. However, the cellular mechanism(s) that enable EBV to establish exclusively lytic infection in normal differentiated oral epithelial cells are not currently understood. Here we show that a cellular transcription factor known to promote epithelial cell differentiation, KLF4, induces differentiation-dependent lytic EBV infection by binding to and activating the two EBV immediate-early gene (BZLF1 and BRLF1) promoters. We demonstrate that latently EBV-infected, telomerase-immortalized normal oral keratinocyte (NOKs) cells undergo lytic viral reactivation confined to the more differentiated cell layers in organotypic raft culture. Furthermore, we show that endogenous KLF4 expression is required for efficient lytic viral reactivation in response to phorbol ester and sodium butyrate treatment in several different EBV-infected epithelial cell lines, and that the combination of KLF4 and another differentiation-dependent cellular transcription factor, BLIMP1, is highly synergistic for inducing lytic EBV infection. We confirm that both KLF4 and BLIMP1 are expressed in differentiated, but not undifferentiated, epithelial cells in normal tongue tissue, and show that KLF4 and BLIMP1 are both expressed in a patient-derived OHL lesion. In contrast, KLF4 protein is not detectably expressed in B cells, where EBV normally enters latent infection, although KLF4 over-expression is sufficient to induce lytic EBV reactivation in Burkitt lymphoma cells. Thus, KLF4, together with BLIMP1, plays a critical role in mediating lytic EBV reactivation in epithelial cells.
The transition from latent Epstein-Barr virus (EBV) infection to lytic viral replication is mediated by the viral transcription factors Rta and Zta. Although both are required for virion production, ...dissecting the specific roles played by Rta and Zta is challenging because they induce each other's expression. To circumvent this, we constructed an EBV mutant deleted for the genes encoding Rta and Zta (BRLF1 and BZLF1, respectively) in the Akata strain BACmid. This mutant, termed EBVΔRZ, was used to infect several epithelial cell lines, including telomerase-immortalized normal oral keratinocytes, a highly physiologic model of EBV epithelial cell infection. Using RNA-seq, we determined the gene expression induced by each viral transactivator. Surprisingly, Zta alone only induced expression of the lytic origin transcripts BHLF1 and LF3. In contrast, Rta activated the majority of EBV early gene transcripts. As expected, Zta and Rta were both required for expression of late gene transcripts. Zta also cooperated with Rta to enhance a subset of early gene transcripts (Rtasynergy transcripts) that Zta was unable to activate when expressed alone. Interestingly, Rta and Zta each cooperatively enhanced the other's binding to EBV early gene promoters, but this effect was not restricted to promoters where synergy was observed. We demonstrate that Zta did not affect Rtasynergy transcript stability, but increased Rtasynergy gene transcription despite having no effect on their transcription when expressed alone. Our results suggest that, at least in epithelial cells, Rta is the dominant transactivator and that Zta functions primarily to support DNA replication and co-activate a subset of early promoters with Rta. This closely parallels the arrangement in KSHV where ORF50 (Rta homolog) is the principal activator of lytic transcription and K8 (Zta homolog) is required for DNA replication at oriLyt.
During the last years, several new routes to produce zeolites with controlled mesoporosity have appeared. Moreover, an improved catalytic performance of the resulting mesoporous zeolites over ...conventional zeolites has been demonstrated in several reactions. In most cases, the mesoporous zeolites exhibit higher catalytic activity, but in some cases also improved selectivity and longer catalyst lifetime has been reported. The beneficial effects of introducing mesopores into the zeolites has in most instances been attributed to improved mass transport to and from the active sites located in the zeolite micropores. Here, we briefly discuss the most important ways of introducing mesopores into zeolites and, for the first time, we show experimentally that the presence of mesopores dramatically increases the rate of diffusion in zeolite catalysts. This is done by studying the elution of
iso-butane from packed beds of conventional and mesoporous zeolite catalysts. Moreover, we discuss in detail the recent observation of improved activity and selectivity in the alkylation of benzene with ethene using mesoporous zeolite single crystal catalysts. For this reaction, we show by calculation of the Thiele modulus that this improved performance can be mainly attributed to a diffusional limitation of ethylbenzene in the zeolite pores. This is verified in new ethylbenzene dealkylation experiments where mesoporous zeolite catalysts show significantly improved activity over conventional zeolite catalysts.
Context:
An important goal during weight loss is to maximize fat loss while preserving metabolically active fat-free mass (FFM). Massive weight loss typically results in substantial loss of FFM ...potentially slowing metabolic rate.
Objective:
Our objective was to determine whether a weight loss program consisting of diet restriction and vigorous exercise helped to preserve FFM and maintain resting metabolic rate (RMR).
Participants and Intervention:
We measured body composition by dual-energy x-ray absorptiometry, RMR by indirect calorimetry, and total energy expenditure by doubly labeled water at baseline (n = 16), wk 6 (n = 11), and wk 30 (n = 16).
Results:
At baseline, participants were severely obese (×± sd; body mass index 49.4 ± 9.4 kg/m2) with 49 ± 5% body fat. At wk 30, more than one third of initial body weight was lost (−38 ± 9%) and consisted of 17 ± 8% from FFM and 83 ± 8% from fat. RMR declined out of proportion to the decrease in body mass, demonstrating a substantial metabolic adaptation (−244 ± 231 and −504 ± 171 kcal/d at wk 6 and 30, respectively, P < 0.01). Energy expenditure attributed to physical activity increased by 10.2 ± 5.1 kcal/kg·d at wk 6 and 6.0 ± 4.1 kcal/kg·d at wk 30 (P < 0.001 vs. zero).
Conclusions:
Despite relative preservation of FFM, exercise did not prevent dramatic slowing of resting metabolism out of proportion to weight loss. This metabolic adaptation may persist during weight maintenance and predispose to weight regain unless high levels of physical activity or caloric restriction are maintained.
Notch1 regulates gene expression by associating with the DNA-binding factor RBPJ and is oncogenic in murine and human T-cell progenitors. Using ChIP-Seq, we find that in human and murine ...T-lymphoblastic leukemia (TLL) genomes Notch1 binds preferentially to promoters, to RBPJ binding sites, and near imputed ZNF143, ETS, and RUNX sites. ChIP-Seq confirmed that ZNF143 binds to ∼40% of Notch1 sites. Notch1/ZNF143 sites are characterized by high Notch1 and ZNF143 signals, frequent cobinding of RBPJ (generally through sites embedded within ZNF143 motifs), strong promoter bias, and relatively low mean levels of activating chromatin marks. RBPJ and ZNF143 binding to DNA is mutually exclusive in vitro, suggesting RBPJ/Notch1 and ZNF143 complexes exchange on these sites in cells. K-means clustering of Notch1 binding sites and associated motifs identified conserved Notch1-RUNX, Notch1-ETS, Notch1-RBPJ, Notch1-ZNF143, and Notch1-ZNF143-ETS clusters with different genomic distributions and levels of chromatin marks. Although Notch1 binds mainly to gene promoters, ∼75% of direct target genes lack promoter binding and are presumably regulated by enhancers, which were identified near MYC, DTX1, IGF1R, IL7R, and the GIMAP cluster. Human and murine TLL genomes also have many sites that bind only RBPJ. Murine RBPJ-only sites are highly enriched for imputed REST (a DNA-binding transcriptional repressor) sites, whereas human RPBJ-only sites lack REST motifs and are more highly enriched for imputed CREB sites. Thus, there is a conserved network of cis-regulatory factors that interacts with Notch1 to regulate gene expression in TLL cells, as well as unique classes of divergent RBPJ-only sites that also likely regulate transcription.
Epstein-Barr virus nuclear antigen 2 (EBNA2) regulation of transcription through the cell transcription factor RBPJ is essential for resting B-lymphocyte (RBL) conversion to immortal lymphoblast cell ...lines (LCLs). ChIP-seq of EBNA2 and RBPJ sites in LCL DNA found EBNA2 at 5,151 and RBPJ at 10,529 sites. EBNA2 sites were enriched for RBPJ (78%), early B-cell factor (EBF, 39%), RUNX (43%), ETS (39%), NFκB (22%), and PU.1 (22%) motifs. These motif associations were confirmed by LCL RBPJ ChIP-seq finding 72% RBPJ occupancy and Encyclopedia Of DNA Elements LCL ChIP-seq finding EBF, NFκB RELA, and PU.1 at 54%, 31%, and 17% of EBNA2 sites. EBNA2 and RBPJ were predominantly at intergene and intron sites and only 14% at promoter sites. K-means clustering of EBNA2 site transcription factors identified RELA-ETS, EBF-RUNX, EBF, ETS, RBPJ, and repressive RUNX clusters, which ranked from highest to lowest in H3K4me1 signals and nucleosome depletion, indicative of active chromatin. Surprisingly, although quantitatively less, the same genome sites in RBLs exhibited similar high-level H3K4me1 signals and nucleosome depletion. The EBV genome also had an LMP1 promoter EBF site, which proved critical for EBNA2 activation. LCL HiC data mapped intergenic EBNA2 sites to EBNA2 up-regulated genes. FISH and chromatin conformation capture linked EBNA2/RBPJ enhancers 428 kb 5' of MYC to MYC. These data indicate that EBNA2 evolved to target RBL H3K4me1 modified, nucleosome-depleted, nonpromoter sites to drive B-lymphocyte proliferation in primary human infection. The primed RBL program likely supports antigen-induced proliferation.
Epstein-Barr virus (EBV)-associated diseases of epithelial cells, including tumors that have latent infection, such as nasopharyngeal carcinoma (NPC), and oral hairy leukoplakia (OHL) lesions that ...have lytic infection, frequently express the viral latent membrane protein 1 (LMP1). In lytically infected cells, LMP1 expression is activated by the BRLF1 (R) immediate early (IE) protein. However, the mechanisms by which LMP1 expression is normally regulated in epithelial cells remain poorly understood, and its potential roles in regulating lytic reactivation in epithelial cells are as yet unexplored. We previously showed that the differentiation-dependent cellular transcription factors KLF4 and BLIMP1 induce lytic EBV reactivation in epithelial cells by synergistically activating the two EBV immediate early promoters (Zp and Rp). Here we show that epithelial cell differentiation also induces LMP1 expression. We demonstrate that KLF4 and BLIMP1 cooperatively induce the expression of LMP1, even in the absence of the EBV IE proteins BZLF1 (Z) and R, via activation of the two LMP1 promoters. Furthermore, we found that differentiation of NOKs-Akata cells by either methylcellulose suspension or organotypic culture induces LMP1 expression prior to Z and R expression. We show that LMP1 enhances the lytic infection-inducing effects of epithelial cell differentiation, as well as 12-
-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate treatment, in EBV-infected epithelial cells by increasing expression of the Z and R proteins. Our results suggest that differentiation of epithelial cells activates a feed-forward loop in which KLF4 and BLIMP1 first activate LMP1 expression and then cooperate with LMP1 to activate Z and R expression.
The EBV protein LMP1 is expressed in EBV-associated epithelial cell diseases, regardless of whether these diseases are due to lytic infection (such as oral hairy leukoplakia) or latent infection (such as nasopharyngeal carcinoma). However, surprisingly little is known about how LMP1 expression is regulated in epithelial cells, and there are conflicting reports about whether it plays any role in regulating viral lytic reactivation. In this study, we show that epithelial cell differentiation induces LMP1 expression by increasing expression of two cellular transcription factors (KLF4 and BLIMP1) which cooperatively activate the two LMP1 promoters. We also demonstrate that LMP1 promotes efficient lytic reactivation in EBV-infected epithelial cells by enhancing expression of the Z and R proteins. Thus, in EBV-infected epithelial cells, LMP1 expression is promoted by differentiation and positively regulates lytic viral reactivation.
Impairments in metabolic flexibility (MF) and substrate handling are associated with metabolic syndrome. However, it is unknown whether metabolic inflexibility causes insulin resistance. We therefore ...measured MF and substrate handling before and after 8 weeks of overfeeding in initially healthy adults as a model of the early stages of insulin resistance.
Twenty-nine healthy men (27±5 years old; body mass index 25.5±2.3 kg m
) were overfed by 40% above baseline energy requirements for 8 weeks and gained 7.6±2.1 kg of weight. Before and after overfeeding, energy expenditure, substrate oxidation and MF were measured in two ways: (a) during 1 day of eucaloric feeding in a whole-room indirect calorimeter and (b) during a two-step hyperinsulinemic-euglycemic clamp.
Eight weeks of overfeeding decreased insulin sensitivity at low and high doses of insulin (P=0.001 and P=0.06, respectively). This was accompanied by decreases in the respiratory quotient (RQ) while sleeping (from 0.877±0.020 to 0.864±0.026; P=0.05) and at low insulin levels during the clamp (from 0.927±0.047 to 0.907±0.032; P=0.01). Overfeeding did not affect MF as measured during a clamp (P⩾0.17), but it tended to increase 24-h MF (awake RQ-sleep RQ) as measured by chamber by 0.010±0.028 (P=0.08). In terms of substrate oxidation, overfeeding increased protein oxidation by 13±23 g day
(P=0.003) and tended to increase fat oxidation by 6±16 g day
(P=0.07) but did not affect carbohydrate oxidation (P=0.64). Individuals with greater metabolic adaptation to overfeeding had higher carbohydrate oxidation rates (r=0.66, P=8 × 10
) but not fat oxidation rates (P=0.09).
The early stages of insulin resistance are accompanied by modest declines in the RQs during sleep and during a clamp, with no changes in fasting RQ or signs of metabolic inflexibility. Our data therefore suggest that metabolic inflexibility does not cause insulin resistance.
Epstein-Barr virus (EBV) is a human herpesvirus that is associated with lymphomas as well as nasopharyngeal and gastric carcinomas. Although carcinomas account for almost 90% of EBV-associated ...cancers, progress in examining EBV's role in their pathogenesis has been limited by difficulty in establishing latent infection in nontransformed epithelial cells. Recently, EBV infection of human telomerase reverse transcriptase (hTERT)-immortalized normal oral keratinocytes (NOKs) has emerged as a model that recapitulates aspects of EBV infection
, such as differentiation-associated viral replication. Using uninfected NOKs and NOKs infected with the Akata strain of EBV (NOKs-Akata), we examined changes in gene expression due to EBV infection and differentiation. Latent EBV infection produced very few significant gene expression changes in undifferentiated NOKs but significantly reduced the extent of differentiation-induced gene expression changes. Gene set enrichment analysis revealed that differentiation-induced downregulation of the cell cycle and metabolism pathways was markedly attenuated in NOKs-Akata relative to that in uninfected NOKs. We also observed that pathways induced by differentiation were less upregulated in NOKs-Akata. We observed decreased differentiation markers and increased suprabasal MCM7 expression in NOKs-Akata versus NOKs when both were grown in raft cultures, consistent with our transcriptome sequencing (RNA-seq) results. These effects were also observed in NOKs infected with a replication-defective EBV mutant (AkataΔRZ), implicating mechanisms other than lytic-gene-induced host shutoff. Our results help to define the mechanisms by which EBV infection alters keratinocyte differentiation and provide a basis for understanding the role of EBV in epithelial cancers.
Latent infection by Epstein-Barr virus (EBV) is an early event in the development of EBV-associated carcinomas. In oral epithelial tissues, EBV establishes a lytic infection of differentiated epithelial cells to facilitate the spread of the virus to new hosts. Because of limitations in existing model systems, the effects of latent EBV infection on undifferentiated and differentiating epithelial cells are poorly understood. Here, we characterize latent infection of an hTERT-immortalized oral epithelial cell line (NOKs). We find that although EBV expresses a latency pattern similar to that seen in EBV-associated carcinomas, infection of undifferentiated NOKs results in differential expression of a small number of host genes. In differentiating NOKs, however, EBV has a more substantial effect, reducing the extent of differentiation and delaying the exit from the cell cycle. This effect may synergize with preexisting cellular abnormalities to prevent exit from the cell cycle, representing a critical step in the development of cancer.
Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network ...properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.
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