Corrigendum: Direct evidence of abortive lytic infection-mediated establishment of Epstein-Barr virus latency during B-cell infection.

[This corrects the article DOI: 10.3389/fmicb.2020.575255.].

Modeling and predicting individual variation in COVID-19 vaccine-elicited antibody response in the general population.

As we learned during the COVID-19 pandemic, vaccines are one of the most important tools in infectious disease control. To date, an unprecedentedly large volume of high-quality data on COVID-19 vaccinations have been accumulated. For preparedness in future pandemics beyond COVID-19, these valuable datasets should be analyzed to best shape an effective vaccination strategy. We are collecting longitudinal data from a community-based cohort in Fukushima, Japan, that consists of 2,407 individuals who underwent serum sampling two or three times after a two-dose vaccination with either BNT162b2 or mRNA-1273. Using the individually reconstructed time courses of the vaccine-elicited antibody response based on mathematical modeling, we first identified basic demographic and health information that contributed to the main features of the antibody dynamics, i.e., the peak, the duration, and the area under the curve. We showed that these three features of antibody dynamics were partially explained by underlying medical conditions, adverse reactions to vaccinations, and medications, consistent with the findings of previous studies. We then applied to these factors a recently proposed computational method to optimally fit an "antibody score", which resulted in an integer-based score that can be used as a basis for identifying individuals with higher or lower antibody titers from basic demographic and health information. The score can be easily calculated by individuals themselves or by medical practitioners. Although the sensitivity of this score is currently not very high, in the future, as more data become available, it has the potential to identify vulnerable populations and encourage them to get booster vaccinations. Our mathematical model can be extended to any kind of vaccination and therefore can form a basis for policy decisions regarding the distribution of booster vaccines to strengthen immunity in future pandemics.

Dose Evaluation in 2-Phase Method for Advanced Esophageal Cancer by Hybrid Irradiation Techniques.

Purpose: In concurrent chemoradiotherapy for advanced esophageal cancer, a 2-phase method consisting of initial irradiation of a wide elective nodal region and boost irradiation of the primary lesion is commonly employed. Although dose escalation to the primary lesion may be required to achieve higher local control rates, the radiation dose to critical organs must not exceed dose constraints. To achieve an optimum balance of dose prescription and dose reduction to surrounding organs, such as the lungs and heart, we compared hybrid dose distributions and investigated the best combination of the following recent irradiation techniques: volumetric modulation arc therapy (VMAT), proton broad-beam irradiation, and intensity-modulated proton beam therapy (IMPT). Materials and Methods: Forty-five patients with advanced esophageal cancer whose primary lesions were located in the middle- or lower-thoracic region were studied. Radiotherapy plans for the initial and boost irradiation in the 2-phase method were calculated using VMAT, proton broad-beam irradiation, and IMPT calculation codes, and the dose-volume histogram indices of the lungs and heart for the accumulated plans were compared. Results: In plans using boost proton irradiation with a prescribed dose of 60 Gy(RBE), all dose-volume histogram indices were significantly below the tolerance limits. Initial and boost irradiation with VMAT resulted in the median dose of V30 Gy(RBE)(heart) of 27.4% and an achievement rate below the tolerance limit of 57.8% (26 cases). In simulations of dose escalation up to 70 Gy(RBE), initial and boost IMPT resulted in the highest achievement rate, satisfying all dose constraints in 95.6% (43 cases). Conclusion: Applying VMAT to both initial and boost irradiation is not recommended because of the increased risk of the cardiac dose exceeding the tolerance limit. IMPT may allow dose escalation of up to 70 Gy(RBE) without radiation risks to the lungs and heart in the treatment of advanced esophageal cancer.

Single-Cell Transcriptomic Analysis of Epstein-Barr Virus-Associated Hemophagocytic Lymphohistiocytosis.

Epstein-Barr virus (EBV) infection can lead to infectious mononucleosis (EBV-IM) and, more rarely, EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), which is characterized by a life-threatening hyperinflammatory cytokine storm with immune dysregulation. Interferon-gamma (IFNγ) has been identified as a critical mediator for primary HLH; however, the detailed role of IFNγ and other cytokines in EBV-HLH is not fully understood. In this study, we used single-cell RNA sequencing to characterize the immune landscape of EBV-HLH and compared it with EBV-IM. Three pediatric patients with EBV-HLH with different backgrounds, one with X-linked lymphoproliferative syndrome type 1 (XLP1), two with chronic active EBV disease (CAEBV), and two patients with EBV-IM were enrolled. The TUBA1B + STMN1 + CD8 + T cell cluster, a responsive proliferating cluster with rich mRNA detection, was explicitly observed in EBV-IM, and the upregulation of SH2D1A-the gene responsible for XLP1-was localized in this cluster. This proliferative cluster was scarcely observed in EBV-HLH cases. In EBV-HLH cases with CAEBV, upregulation of LAG3 was observed in EBV-infected cells, which may be associated with an impaired response by CD8 + T cells. Additionally, genes involved in type I interferon (IFN) signaling were commonly upregulated in each cell fraction of EBV-HLH, and activation of type II IFN signaling was observed in CD4 + T cells, natural killer cells, and monocytes but not in CD8 + T cells in EBV-HLH. In conclusion, impaired responsive proliferation of CD8 + T cells and upregulation of type I IFN signaling were commonly observed in EBV-HLH cases, regardless of the patients' background, indicating the key features of EBV-HLH.

Epstein-Barr virus lytic gene BNRF1 promotes B-cell lymphomagenesis via IFI27 upregulation.

Epstein-Barr virus (EBV) is a ubiquitous human lymphotropic herpesvirus that is causally associated with several malignancies. In addition to latent factors, lytic replication contributes to cancer development. In this study, we examined whether the lytic gene BNRF1, which is conserved among gamma-herpesviruses, has an important role in lymphomagenesis. We found that lymphoblastoid cell lines (LCLs) established by BNRF1-knockout EBV exhibited remarkably lower pathogenicity in a mice xenograft model than LCLs produced by wild-type EBV (LCLs-WT). RNA-seq analyses revealed that BNRF1 elicited the expression of interferon-inducible protein 27 (IFI27), which promotes cell proliferation. IFI27 knockdown in LCLs-WT resulted in excessive production of reactive oxygen species, leading to cell death and significantly decreased their pathogenicity in vivo. We also confirmed that IFI27 was upregulated during primary infection in B-cells. Our findings revealed that BNRF1 promoted robust proliferation of the B-cells that were transformed by EBV latent infection via IFI27 upregulation both in vitro and in vivo.

Suspension Concentrate crop protection formulation design and performance for low spray volume and UAS spray application.

BACKGROUND: Unmanned aerial systems (UAS) are providing interesting disruptive solutions for spray application of crop protection products with very-low spray volumes (VLV) down to 8 L/ha that offer improved sustainability through reduced water volumes and reduced soil compaction. However, the efficacy of products can be reduced by the significantly lower crop/plant spray coverage and formulation designs that can compensate for this are highly important here. RESULTS: Suspension Concentrate (SC) formulations designed for VLV use containing and delivering low dose rates (g/ha) of organosilicone alkoxylate high-spreading surfactants were found to result in leaf coverage of VLVs comparable to those observed at higher spray volumes. High spreading was observed on textured leaf surfaces containing sub-micron sized epicuticular wax crystals. Greenhouse fungal disease studies showed enhanced efficacy with these SC formulations compared to standard SC formulations without these additives and maintained the observed increase in efficacy when applied at VLV. Alternatively, SC formulations without high spreading formulants but containing uptake promoting nonionic surfactants showed enhanced cuticle penetration through isolated cuticles at VLV in comparison to higher spray volumes, with coffee-ring spray deposit microstructures present at VLVs. Similarly, greenhouse studies showed enhanced efficacy that was maintained at VLV relative to SCs without these additives. CONCLUSION: At VLVs, SC formulations applied at relatively low dose rates (g/ha) of formulants (adjuvants) enhancing spreading on the leaf surface and/or uptake of the active ingredient(s) maintained good spreading, uptake and biological efficacy in greenhouse studies overcoming the coverage limitations of SC formulations without these additives. This result is unexpected considering the low dose rate of adjuvants used. © 2023 Bayer AG. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Generalized Verrucosis on a Background of 3C Syndrome Treated With Subcutaneous IgG Supplementation.

This case report describes a man in his 20s with 3C/Ritscher-Schinzel syndrome who presented to the dermatology department after developing multiple skin lesions.

Growth Transformation of B Cells by Epstein-Barr Virus Requires IMPDH2 Induction and Nucleolar Hypertrophy.

The in vitro growth transformation of primary B cells by Epstein-Barr virus (EBV) is the initial step in the development of posttransplant lymphoproliferative disorder (PTLD). We performed electron microscopic analysis and immunostaining of primary B cells infected with wild-type EBV. Interestingly, the nucleolar size was increased by two days after infection. A recent study found that nucleolar hypertrophy, which is caused by the induction of the IMPDH2 gene, is required for the efficient promotion of growth in cancers. In the present study, RNA-seq revealed that the IMPDH2 gene was significantly induced by EBV and that its level peaked at day 2. Even without EBV infection, the activation of primary B cells by the CD40 ligand and interleukin-4 increased IMPDH2 expression and nucleolar hypertrophy. Using EBNA2 or LMP1 knockout viruses, we found that EBNA2 and MYC, but not LMP1, induced the IMPDH2 gene during primary infections. IMPDH2 inhibition by mycophenolic acid (MPA) blocked the growth transformation of primary B cells by EBV, leading to smaller nucleoli, nuclei, and cells. Mycophenolate mofetil (MMF), which is a prodrug of MPA that is approved for use as an immunosuppressant, was tested in a mouse xenograft model. Oral MMF significantly improved the survival of mice and reduced splenomegaly. Taken together, these results indicate that EBV induces IMPDH2 expression through EBNA2-dependent and MYC-dependent mechanisms, leading to the hypertrophy of the nucleoli, nuclei, and cells as well as efficient cell proliferation. Our results provide basic evidence that IMPDH2 induction and nucleolar enlargement are crucial for B cell transformation by EBV. In addition, the use of MMF suppresses PTLD. IMPORTANCE EBV infections cause nucleolar enlargement via the induction of IMPDH2, which are essential for B cell growth transformation by EBV. Although the significance of IMPDH2 induction and nuclear hypertrophy in the tumorigenesis of glioblastoma has been reported, EBV infection brings about the change quickly by using its transcriptional cofactor, EBNA2, and MYC. Moreover, we present here, for the novel, basic evidence that an IMPDH2 inhibitor, namely, MPA or MMF, can be used for EBV-positive posttransplant lymphoproliferative disorder (PTLD).

Generation and purification of ACTH-secreting hPSC-derived pituitary cells for effective transplantation.

Pituitary organoids are promising graft sources for transplantation in treatment of hypopituitarism. Building on development of self-organizing culture to generate pituitary-hypothalamic organoids (PHOs) using human pluripotent stem cells (hPSCs), we established techniques to generate PHOs using feeder-free hPSCs and to purify pituitary cells. The PHOs were uniformly and reliably generated through preconditioning of undifferentiated hPSCs and modulation of Wnt and TGF-ß signaling after differentiation. Cell sorting using EpCAM, a pituitary cell-surface marker, successfully purified pituitary cells, reducing off-target cell numbers. EpCAM-expressing purified pituitary cells reaggregated to form three-dimensional pituitary spheres (3D-pituitaries). These exhibited high adrenocorticotropic hormone (ACTH) secretory capacity and responded to both positive and negative regulators. When transplanted into hypopituitary mice, the 3D-pituitaries engrafted, improved ACTH levels, and responded to in vivo stimuli. This method of generating purified pituitary tissue opens new avenues of research for pituitary regenerative medicine.

Epstein-Barr Virus BBLF1 Mediates Secretory Vesicle Transport to Facilitate Mature Virion Release.

Enveloped viruses undergo a complex multistep process of assembly, maturation, and release into the extracellular space utilizing host secretory machinery. Several studies of the herpesvirus subfamily have shown that secretory vesicles derived from the trans-Golgi network (TGN) or endosomes transport virions into the extracellular space. However, the regulatory mechanism underlying the release of Epstein-Barr virus, a human oncovirus, remains unclear. We demonstrate that disruption of BBLF1, a tegument component, suppressed viral release and resulted in the accumulation of viral particles on the inner side of the vesicular membrane. Organelle separation revealed the accumulation of infectious viruses in fractions containing vesicles derived from the TGN and late endosomes. Deficiency of an acidic amino acid cluster in BBLF1 reduced viral secretion. Moreover, truncational deletion of the C-terminal region of BBLF1 increased infectious virus production. These findings suggest that BBLF1 regulates the viral release pathway and reveal a new aspect of tegument protein function. IMPORTANCE Several viruses have been linked to the development of cancer in humans. Epstein-Barr virus (EBV), the first identified human oncovirus, causes a wide range of cancers. Accumulating literature has demonstrated the role of viral reactivation in tumorigenesis. Elucidating the functions of viral lytic genes induced by reactivation, and the mechanisms of lytic infection, is essential to understanding pathogenesis. Progeny viral particles synthesized during lytic infection are released outside the cell after the assembly, maturation, and release steps, leading to further infection. Through functional analysis using BBLF1-knockout viruses, we demonstrated that BBLF1 promotes viral release. The acidic amino acid cluster in BBLF1 was also important for viral release. Conversely, mutants lacking the C terminus exhibited more efficient virus production, suggesting that BBLF1 is involved in the fine-tuning of progeny release during the EBV life cycle.

Longer Survival and Preserved Liver Function after Proton Beam Therapy for Patients with Unresectable Hepatocellular Carcinoma.

BACKGROUND: Proton beam therapy (PBT) has been recently reported to achieve excellent tumor control with minimal toxicity in patients with unresectable hepatocellular carcinoma (HCC). Radiofrequency ablation (RFA) combined with transcatheter arterial chemoembolization (TACE) was investigated for larger HCC. This study was designed to evaluate the therapeutic effect of PBT on unresectable HCC in comparison with TACE combined with RFA. METHODS: We retrospectively analyzed 70 patients with HCC which was difficult to control by surgical resection or RFA monotherapy, 24 patients treated with PBT and 46 patients with TACE plus RFA. The therapeutic effects were assessed as local progression-free survival (PFS) and overall survival (OS). RESULTS: The local PFS was more than 65% in 60 months for PBT and TACE plus RFA. The patients treated with PBT showed 82% OS at 60 months post-treatment. In contrast, those treated with TACE plus RFA showed 28% OS. When comparing the changes of ALBI scores in patients with different severities of chronic liver disease, the scores of PBT-treated patients were maintained at the baseline; however, those of TACE plus RFA-treated patients worsened after the treatments. CONCLUSIONS: The results indicated that PBT may show better benefits than TACE plus RFA therapy in terms of OS in patients with unresectable HCC by sparing the non-tumor liver tissues.

A Japanese registry study and systematic review of particle therapy for renal cell carcinoma.

The feasibility and efficacy of particle beam therapy (PBT) using protons or carbon ions were compared with those of photon-based stereotactic body radiotherapy (SBRT) for primary renal cell carcinoma (RCC) via a systematic review and nationwide registry for PBT (Japanese Society for Radiation Oncology [JASTRO] particle therapy committee). Between July 2016 and May 2019, 20 patients with non-metastatic RCC who were treated at six Japanese institutes (using protons at three, using carbon ions at the other three) were registered in the nationwide database and followed up prospectively. The 20 patients comprised 15 men and had a median age of 67 (range: 57-88) years. The total radiation dose was 66-79.6 Gy (relative biological effectiveness [RBE]). Over a median follow up of 31 months, the 3-year rates of overall survival (OS) and local control (LC) were 100% and 94.4%, respectively. No grade ≥ 3 toxicities were observed. Based on a random effects model, a meta-analysis including the present results revealed 3-year OS rates after SBRT and PBT of 75.3% (95% CI: 57.3-86.6) and 94.3% (95% CI: 86.8-97.6), respectively (P = 0.005), but the difference in LC rates between the two methods was not observed (P = 0.63). PBT is expected to have similar if not better treatment results compared with SBRT for primary renal cancer. In particular, PBT was shown to be effective even for large RCC and could provide a therapeutic option when SBRT is not indicated.

Effectiveness of CT-image guidance in proton therapy for liver cancer and the importance of daily dose monitoring for tumors and organs at risk.

BACKGROUND: It is important to have precise image guidance throughout proton therapy in order to take advantage of the therapy's physical selectivity. PURPOSE: We evaluated the effectiveness of computed tomography (CT)-image guidance in proton therapy for patients with hepatocellular carcinoma (HCC) by assessing daily proton dose distributions. The importance of daily CT image-guided registration and daily proton dose monitoring for tumors and organs at risk (OARs) was investigated. METHODS: A retrospective analysis was conducted using 570 sets of daily CT (dCT) images throughout whole treatment fractions for 38 HCC patients who underwent passive scattering proton therapy with either a 66 cobalt gray equivalent (GyE)/10 fractions (n = 19) or 76 GyE/20 fractions (n = 19) protocol. The actual daily delivered dose distributions were estimated by forward calculation using the dCT sets, their corresponding treatment plans, and the recorded daily couch correction information. We then evaluated the daily changes of the dose indices D99% , V30GyE , and Dmax for the tumor volumes, non-tumorous liver, and other OARs, that is, stomach, esophagus, duodenum, colon, respectively. Contours were created for all dCT sets. We validated the efficacy of the dCT-based tumor registrations (hereafter, "tumor registration") by comparing them with the bone registration and diaphragm registration as a simulation of the treatment based on the positioning using the conventional kV X-ray imaging. The dose distributions and the indices of three registrations were obtained by simulation using the same dCT sets. RESULTS: In the 66 GyE/10 fractions, the daily D99% value in both the tumor and diaphragm registrations agreed with the planned value with 3%-6% (SD), and the V30GyE value for the liver agreed within ±3%; the indices in the bone registration showed greater deterioration. Nevertheless, tumor-dose deterioration occurred in all registration methods for two cases due to daily changes of body shape and respiratory condition. In the 76 GyE/20 fractions, in particular for such a treatment that the dose constraints for the OARs have to be cared in the original planning, the daily D99% in the tumor registration was superior to that in the other registration (p < 0.001), indicating the effectiveness of the tumor registration. The dose constraints, set in the plan as the maximum dose for OARs (i.e., duodenum, stomach, colon, and esophagus) were maintained for 16 patients including seven treated with re-planning. For three patients, the daily Dmax increased gradually or changed randomly, resulting in an inter-fractional averaged Dmax higher than the constraints. The dose distribution would have been improved if re-planning had been conducted. The results of these retrospective analyses indicate the importance of daily dose monitoring followed by adaptive re-planning when needed. CONCLUSIONS: The tumor registration in proton treatment for HCC was effective to maintain the daily dose to the tumor and the dose constraints of OARs, particularly in the treatment where the maintenance for the dose constraints needs to be considered throughout the treatment. Nevertheless daily proton dose monitoring with daily CT imaging is important for more reliable and safer treatment.

Evaluation of Exposure Doses of Elective Nodal Irradiation in Chemoradiotherapy for Advanced Esophageal Cancer.

We evaluated elective nodal irradiation (ENI) doses during radical chemoradiotherapy (CRT) for esophageal cancer (EC). A total of 79 patients (65 men and 14 women) aged 52-80 years with T1-3, N0-3, and M0 (including M1ly) who underwent CRT for EC during November 2012-September 2019 were eligible for this retrospective analysis. Patients were divided into two groups: the high-dose group (HG), including 38 patients who received ≥40 Gy as ENI; and the low-dose group (LG), including 41 patients who received <40 Gy. The median doses were 40.0 and 36.0 Gy in HG and LG, respectively. During the follow-up (median: 36.7 months), no lymph node recurrence was observed in the ENI field in all patients. Lymph node recurrence near the ENI field was observed in six patients. No significant differences were observed between the two groups in median overall survival, progression-free survival, and local control. Grade 3-4 acute and late adverse events were observed in five patients of HG and six patients of LG, respectively. No ulceration or stricture was observed in the ENI field on endoscopy examined with 58 Gy irradiation. In conclusion, an ENI dose of 36 Gy could be considered to control the elective nodes of EC.

Prevalence of suspected autism spectrum disorder and attention-deficit hyperactivity disorder in a Japanese clinical sample with gambling disorder: A cross-sectional study.

Aim: Studies show gambling disorders are associated with attention-deficit hyperactivity disorder (ADHD). The association between gambling disorders and autism spectrum disorder (ASD) has not been well studied, although ASD is often comorbid with ADHD and is associated with gaming disorder. The aim of this study was to estimate the prevalence of ASD and ADHD traits comorbid with gambling disorders and to examine the relationships between these traits and gambling problems in a clinical population. Methods: This single-site cross-sectional study was conducted at a Japanese addiction outpatient clinic treating gambling disorders. The Autism-Spectrum Quotient (AQ) test and the Adult ADHD Self-Report Scale (ASRS) were used to screen ASD and ADHD. The Problem Gambling Severity Index (PGSI) was used to assess the severity of the gambling problems. We calculated the prevalence of suspected ASD and ADHD with 95% confidence intervals (CI) based on a binomial distribution and performed univariate analyses to examine the relationships between the AQ and ASRS scores and the total PGSI score. Results: We included 97 of 197 potential participants. After screening the participants using the AQ and ASRS, we found that the prevalence of ASD traits was 29.8% (95% CI: 21.0%-40.2%), while the prevalence of ADHD traits was 26.0% (95% CI: 17.9%-36.2%). Univariate regression analyses revealed that the total AQ score was inversely associated with the total PGSI score. However, the total ASRS score and some ASRS subscores were positively associated with the total PGSI score. Conclusion: ASD and ADHD may be prevalent among patients with gambling disorders in clinical settings.

Long-Term Results of Proton Therapy for Hepatocellular Carcinoma Using Four-Dimensional Computed Tomography Planning without Fiducial Markers.

We report here the long-term results of marker-less respiratory-gated proton therapy (PT), without fiducial markers for hepatocellular carcinoma (HCC), which was planned using a four-dimensional computed tomography technique. Local tumor control (LTC) and overall survival (OS) were estimated using the Kaplan-Meier method. Toxicity was graded per CTCAE v5.0. Patients (n = 105; median age 73 years, range 38-90 years) with 128 lesions were treated. The median radiation dose was 66 gray relative biological effectiveness (GyRBE) (range, 52.8-82.5 GyRBE) delivered in 2.0 to 6.6 GyRBE fractions, depending on lesion volume, the involved liver, and the patient's condition. The median follow-up of surviving patients was 63 months (range, 1-126 months), and the 5-year LTC and OS rates were 93.2% and 40.4%, respectively. Univariate and multivariate analyses identified tumors near the gastrointestinal tract as an independent risk factor for local recurrence and revealed that hepatic reserve, tumor stage, performance status, operability, sex, and portal vein thrombosis were independent risk factors for OS. Acute and late treatment-related grade 3 toxicities were experienced by eight patients (7.6%). Adverse events ≥ grade 4 were not evident. Marker-less respiratory-gated PT for HCC is a safe and effective treatment without severe complications.

The Epstein-Barr Virus Enhancer Interaction Landscapes in Virus-Associated Cancer Cell Lines.

Epstein-Barr virus (EBV) persists in human cells as episomes. EBV episomes are chromatinized and their 3D conformation varies greatly in cells expressing different latency genes. We used HiChIP, an assay which combines genome-wide chromatin conformation capture followed by deep sequencing (Hi-C) and chromatin immunoprecipitation (ChIP), to interrogate the EBV episome 3D conformation in different cancer cell lines. In an EBV-transformed lymphoblastoid cell line (LCL) GM12878 expressing type III EBV latency genes, abundant genomic interactions were identified by H3K27ac HiChIP. A strong enhancer was located near the BILF2 gene and looped to multiple genes around BALFs loci. Perturbation of the BILF2 enhancer by CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) altered the expression of BILF2 enhancer-linked genes, including BARF0 and BALF2, suggesting that this enhancer regulates the expression of linked genes. H3K27ac ChIP followed by deep sequencing (ChIP-seq) identified several strong EBV enhancers in T/NK (natural killer) lymphoma cells that express type II EBV latency genes. Extensive intragenomic interactions were also found which linked enhancers to target genes. A strong enhancer at BILF2 also looped to the BALF loci. CRISPRi also validated the functional connection between BILF2 enhancer and BARF1 gene. In contrast, H3K27ac HiChIP found significantly fewer intragenomic interactions in type I EBV latency gene-expressing primary effusion lymphoma (PEL) cell lines. These data provided new insight into the regulation of EBV latency gene expression in different EBV-associated tumors. IMPORTANCE EBV is the first human DNA tumor virus identified, discovered over 50 years ago. EBV causes ~200,000 cases of various cancers each year. EBV-encoded oncogenes, noncoding RNAs, and microRNAs (miRNAs) can promote cell growth and survival and suppress senescence. Regulation of EBV gene expression is very complex. The viral C promoter regulates the expression of all EBV nuclear antigens (EBNAs), some of which are very far away from the C promoter. Another way by which the virus activates remote gene expression is through DNA looping. In this study, we describe the viral genome looping patterns in various EBV-associated cancer cell lines and identify important EBV enhancers in these cells. This study also identified novel opportunities to perturb and eventually control EBV gene expression in these cancer cells.

EBV Exploits RNA m6A Modification to Promote Cell Survival and Progeny Virus Production During Lytic Cycle.

N6-methyladenosine (m6A) mediates various biological processes by affecting RNA stability, splicing, and translational efficiency. The roles of m6A modification in Epstein-Barr virus (EBV) infection in the lytic phase are unclear. Here, knockout of the m6A methyltransferase, N6-methyladenosine methyltransferase-like 3 (METTL3), or inhibition of methylation by DAA or UZH1a decreased the expression of viral lytic proteins and reduced progeny virion production. Interestingly, cell growth and viability were decreased by induction of the lytic cycle in METTL3-knockout or inhibitor-treated cells. Apoptosis was induced in those conditions possibly because of a decreased level of the anti-apoptotic viral protein, BHRF1. Therefore, m6A shows potential as a target of lytic induction therapy for EBV-associated cancers, including Burkitt lymphoma.

Direct reprogramming of adult adipose-derived regenerative cells toward cardiomyocytes using six transcriptional factors.

It is widely accepted that adipose-derived regenerative cells (ADRCs) can differentiate into mesodermal lineage cells. However, reprogramming adult ADRCs into mature cardiomyocytes is challenging. We investigated the induction of myocardial differentiation in ADRCs via direct reprogramming using lentiviral gene transfer. First, we identified candidate transcriptional factors by performing RNA sequencing and ultimately confirmed that the combination of six unique factors (Baf60c, Gata4, Gata6, Klf15, Mef2a, and Myocd) could efficiently express enhanced green fluorescent protein (GFP) in ADRCs isolated from adult alpha-myosin heavy chain promoter-driven GFP transgenic mice. The GFP-positive ADRCs induced by six factors (6F-ADRCs) expressed multiple cardiac genes and revealed cardiac differentiation in bioinformatic analysis. Moreover, injection of 6F-ADRCs into acute myocardial infarcted tissues in vivo resulted in the improvement of survival rate, fractional shortening, and reduction of infarction scar area. This study provides an alternative method for direct reprogramming of adult ADRCs into cardiomyocytes.