The long‐lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein‐Barr virus

The Epstein‐Barr gammaherpesvirus (EBV) is the first oncogenic virus discovered in human. Indeed, EBV has been known for more than 50 years to be tightly associated with certain human cancers. As such, EBV has been the subject of extensive studies aiming at deciphering various aspects of its biological cycle, ranging from the regulation of its genome replication and maintenance to the induction of its lytic cycle, including the mechanisms that allow its immune evasion or that are related to its tumorogenicity. For more than 30 years the budding yeast Saccharomyces cerevisiae has fruitfully contributed to a number of these studies. The aim of this article is to review the various aspects of EBV biology for which yeast has been instrumental, and to propose new possible applications for these yeast‐based assays, as well as the creation of further yeast models dedicated to EBV. This review article illustrates the tremendous potential of S. cerevisiae in integrated chemobiological approaches for the biomedical research.     

TLR3/TRIF signalling pathway regulates IL‐32 and IFN‐β secretion through activation of RIP‐1 and TRAF in the human cornea

Toll‐like receptor‐3 (TLR3) and RNA helicase retinoic‐acid‐inducible protein‐1 (RIG‐I) serve as cytoplasmic sensors for viral RNA components. In this study, we investigated how the TLR3 and RIG‐I signalling pathway was stimulated by viral infection to produce interleukin (IL)‐32‐mediated pro‐inflammatory cytokines and type I interferon in the corneal epithelium using Epstein–Barr virus (EBV)‐infected human cornea epithelial cells (HCECs/EBV) as a model of viral keratitis. Increased TLR3 and RIG‐I that are responded to EBV‐encoded RNA 1 and 2 (EBER1 and EBER2) induced the secretion of IL‐32‐mediated pro‐inflammatory cytokines and IFN‐β through up‐regulation of TRIF/TRAF family proteins or RIP‐1. TRIF silencing or TLR3 inhibitors more efficiently inhibited sequential phosphorylation of TAK1, TBK1, NF‐κB and IRFs to produce pro‐inflammatory cytokines and IFN‐β than RIG‐I‐siRNA transfection in HCECs/EBV. Blockade of RIP‐1, which connects the TLR3 and RIG‐I pathways, significantly blocked the TLR3/TRIF‐mediated and RIG‐I‐mediated pro‐inflammatory cytokines and IFN‐β production in HCECs/EBV. These findings demonstrate that TLR3/TRIF‐dependent signalling pathway against viral RNA might be a main target to control inflammation and anti‐viral responses in the ocular surface.     

Serous effusion cytology of extranodal natural killer/T-cell lymphoma

X.‐Y. Su, J. Huang, Y. Jiang, Y. Tang, G.‐D. Li and W.‐P. Liu Serous effusion cytology of extranodal natural killer/T‐cell lymphoma Objective: Extranodal natural killer/T‐cell lymphoma, nasal type (ENKTCL‐N), is a rare form of lymphoma that typically occurs at extranodal sites. It is one of the most common extranodal lymphomas in China. Literature on effusions and cytological findings relating to ENKTCL‐N is limited. We studied five consecutive cases of ENKTCL‐N effusions collected over a 3‐year period. The cytomorphological, immunocytochemical and molecular biological features were evaluated with literature review. The purpose of this study is to discuss how to diagnose ENKTCL‐N cytologically in effusions. Methods: Smears and cell block sections were reviewed for each case. Immunocytochemistry was performed on 4‐μm paraffin sections. Antibodies used were as follows: cCD3 (intracytoplasmic CD3), CD45RO, surface CD3, CD20, CD79a, CD56, TIA‐1, granzyme B, CD30, CD99, TdT and Ki‐67. In situ hybridization for EBER1/2 (EBER‐ISH) and T‐cell receptor γ (TCRγ) gene rearrangement were performed for all cases. Results: Large to medium‐sized tumour cells with pleomorphic nuclei and coarse chromatin were found in a necrotic background in all cases. The cytoplasm of the tumour cells was scant to moderately abundant with occasional cytoplasmic projections; in Giemsa‐stained smears, fine granules were present in some tumour cells. Mitotic figures were frequent. The tumour cells were all positive for CD56, granzyme B, TIA‐1 and cCD3, and were negative for surface CD3, CD20 or CD79a, CD99 and TdT. The MIB index was 50–80%. Epstein‐Barr virus‐encoded RNA (EBER) hybridizing signals were detected for most neoplastic cells. The T‐cell receptor gamma gene rearrangement analysis showed germ‐line configuration, except for one case. Conclusions: Effusion cytology may be appropriate for establishing the diagnosis of ENKTCL‐N, particularly for patients in whom tissue biopsy is not possible.     

巴氏小体案例在遗传学教学中的应用

细胞遗传学在染色体水平上有3个经典问题,即巴氏小体、多线染色体和灯刷染色体的形成机制和遗传学效应。其中巴氏小体因其与哺乳动物在两性间X染色体的剂量补偿效应、人类性别鉴定和某些人类疾病的相关性而引起科研和教学工作者的持续关注。在遗传学教学过程中,作者尝试将国外的案例教学方式引入教学实践,将巴氏小体这一经典遗传学问题作为一条线贯穿于遗传学教学的部分环节,例如伴性遗传、基因表达调控、癌症发生以及遗传学实验,最后通过课堂讨论会的形式全面总结相关的遗传学知识。结果发现,这种改进的教学方法不仅可以优化遗传学教学内容,拓宽并巩固学生的遗传学基础知识,形成了对一个经典遗传学问题的系统观、发展观;还能引导和激发学生对生命科学的兴趣,收到了良好的教学效果。     

Down-regulation of proteolytic complexes following EBV activation in BL cells

In Burkitt's lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin-proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the alpha/beta subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6' subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.     

Deciphering nasopharyngeal carcinoma pathogenesis via proteomics

Introduction: Nasopharyngeal carcinoma (NPC) is a distinct head and neck squamous cell carcinoma in its etiological association of Epstein–Barr virus (EBV) infection, hidden anatomical location, remarkable racial and geographical distribution, and high incidence of locoregional recurrence or metastasis. Thanks to the advancements in proteomics in recent decades, more understanding of the disease etiology, carcinogenesis, and progression has been gained, potentially deciphering the molecular characteristics of the malignancy.

Areas covered: In this review, we provide an overview of the proteomic aberrations that are likely involved or drive NPC development and progression, focusing on the contributions of major EBV-encoded factors, intercommunication with environment, protein features of high metastasis and therapy resistance, and protein–protein interactions that allow NPC cells to evade immune recognition and elimination. Finally, multistep carcinogenesis and subtypes of NPC from a proteomic perspective are inquired.

Expert commentary: Proteomic studies have covered various aspects involved in NPC pathogenesis, yet much remains to be uncovered. Coherent study designs, optimal conditions for obtaining high-quality data, and compelling interpretation are critical in ensuring the emergence of good science out of NPC proteomics. NPC proteogenomics and proteoform analysis are two promising fields to promote the application of the proteomic findings from bench to bedside.     

NK cell lymphoma, nasal type, with massive lung involvement: a case report

Extranodal NK/T cell lymphoma, nasal type, is an Epstein–Barr virus-associated lymphoma that most commonly involves the nasal cavity and upper respiratory tract. Lung involvement by NK/T cell lymphoma is rare and seldom reported in the literature. We describe the unusual case of a 41-year-old male with NK cell lymphoma, nasal type, who presented with massive secondary lung involvement 2.5 years after the detection of a retroperitoneal mass. The diagnosis was made by open lung biopsy. Despite aggressive treatment, the patient died shortly after the initiation of therapy. Lung involvement by NK/T cell lymphoma occurs most commonly as part of widely disseminated disease and carries a poor prognosis for the patient. Novel agents and innovative therapies need to be developed for this aggressive lymphoma.     

The effect of 1-deoxymannojirimycin on rat liver alpha-mannosidases

Cloned murine mammary tumor virus (MuMTV) sequences allowed us to search for murine mammary tumor virus related sequences in the DNA of surgically removed human breast tumors. Out of 28 tumors so far examined two were found to contain an Eco RI DNA fragment homologous to the long terminal repeat-group antigen (LTR-Gag) and the Envelope (Env) sequences of MuMTV. We have taken the lymphocytes of these patients and cultured them. Rapid growth of lymphocytes, mostly of T origin, occurred in the presence of T cell growth factor (TCGF). Whereas DNA extracted from fresh lymphocytes is negative, that extracted from the 3-day cultured lymphocytes showed MuMTV related sequences. Long term cultures of T cells and a similar culture derived from a healthy person donor were negative at all stages. DNA extracted from the Ebstein Barr Virus-transformed B cells of the patient does not contain the MuMTV related sequences.     

Expression of wild-type and mutant medium-chain acyl-CoA dehydrogenase (MCAD) cDNA in eucaryotic cells

An effective EBV-based expression system for eucaryotic cells has been developed and used for the study of the mitochondrial enzyme medium-chain acyl-CoA dehydrogenase (MCAD). 1325 bp of PCR-generated cDNA, containing the entire coding region, was placed between the SV40 early promotor and polyadenylation signals in the EBV-based vector. Both wild-type MCAD cDNA and cDNA containing the prevalent disease-causing mutation A to G at position 985 of the MCAD cDNA were tested. In transfected COS-7 cells, the steady state amount of mutant MCAD protein was consistently lower than the amount of wild-type human enzyme. The enzyme activity in extracts from cells harbouring the wild-type MCAD cDNA was dramatically higher than in the controls (harbouring the vector without the MCAD gene) while only a slightly higher activity was measured with the mutant MCAD. The mutant MCAD present behaves like wild-type MCAD with respect to solubility, subcellular location, mature protein size and tetrameric structure. In immunoblot comparisons, the MCAD protein was present in normal fibroblasts, but essentially undetectable in patient fibroblasts homozygous for the prevalent mutation. We suggest that the MCAD protein carrying this mutation has an impaired ability to form correct tetramers, leading to instability and subsequent degradation of the enzyme. This finding is discussed in relation to the results from expression of human MCAD in Escherichia coli, where preliminary results show that production of mutant MCAD leads to the formation of aggregates.