A novel and rapid method to quantify cytolytic replication of picornaviruses in cell culture

Determining viral titers is a key issue in a wide variety of studies regarding different aspects of virology. The standard methods used for determining picornavirus titers are endpoint titration assay and plaque assay, both time consuming and laborious. The method described uses the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) that is reduced to formazane by cellular dehydrogenase, genes shown to be down-regulated during picornavirus infection. The amount formazane produced correlates with the viral titers obtained and can easily be measured using an ELISA plate reader. The colorimetric method has been evaluated using virus types from different genera of the Picornaviridae family. The MTT method reduces the time spent on determining the viral titers and still maintains a reliable accuracy.     

Low molecular weight fluorescent probes with good photostability for imaging RNA-rich nucleolus and RNA in cytoplasm in living cells

We have synthesized two low molecular weight organic molecules, PY and IN successfully, which selectively stain nucleolus and cytoplasm of living cells in 30 min, with a much lower uptake in the nucleus. Nucleic acids electrophoresis and digest test of ribonuclease indicate their markedly higher affinity for RNA, especially PY. Moreover their RNA localization in cells is further supported by digest test of ribonuclease, namely, the nucleolar fluorescence signal is distinctly lost upon treatment with RNase. And, the fact that live cells stained by PY and IN still possess physiological function can be confirmed: 1) MTT assay demonstrates that the mitochondria of cells stained remains its electron mediating ability, 2) Double assay of PY/IN and propidium iodide as well as trypan blue testing show that the membrane of cells stained still is intact. Importantly, compared with the only commercial RNA probe, SYTO RNA-Select, PY and IN exhibit much better photostability when continuously illuminated with 488 nm laser and mercury lamp. These results prove that PY and IN are very attractive staining reagents for visualizing RNA in living cells.     

Deletions within the 5'UTR of coxsackievirus B3: consequences for virus translation and replication

Key features of an ideal RNA-based vaccine against coxsackievirus B3 (CVB3) are (i) limited genome replication/virus production (to minimize vaccine-related pathology) and (ii) abundant virus protein synthesis (to maximize immunogenicity). These attributes may apply to CVB3 RNAs lacking up to 250 nucleotides (nt) from their 5' terminus; these RNAs do not give rise to infectious progeny, but they have been reported to retain the entire CVB3 IRES (mapped to nt approximately 432-639) and to produce large quantities of viral protein in transfected cells. Here, we constructed five 5' RNA deletion variants that, to our surprise, failed to protect against CVB3 challenge. We investigated the reasons for this failure and conclude that (i) a 5' terminal deletion as short as 32 nt abolishes CVB3 RNA replication in transfected cells; (ii) this deleted RNA, and others with longer deletions, do not direct abundant protein synthesis in transfected cells, probably as a consequence of their replicative incapacity; and (iii) the CVB3 IRES is substantially larger than previously thought, and its 5' boundary lies between residues 76 and 125, very closely approximating that of the poliovirus IRES.     

Structural analysis provides insights into the modular organization of picornavirus IRES

Picornavirus RNA translation is driven by the internal ribosome entry site (IRES) element. The impact of RNA structure on the foot-and-mouth disease virus (FMDV) IRES activity has been analyzed using Selective 2'Hydroxyl Acylation analyzed by Primer Extension (SHAPE) and high throughput analysis of RNA conformation by antisense oligonucleotides printed on microarrays. SHAPE reactivity revealed the self-folding capacity of domain 3 and evidenced a change of RNA structure in a defective GNRA mutant. A modified RNA conformation of this mutant was also evidenced by RNA accessibility to oligonucleotides. Interestingly, comparison of nucleotide reactivity with RNA accessibility revealed that SHAPE reactive nucleotides corresponding to the GNRA motif were not accessible to their respective target oligonucleotides. The differential response was observed both in domain 3 and the entire IRES. Our results demonstrate distant effects of the GNRA motif in the domain 3 RNA conformation, and highlight the modular organization of a picornavirus IRES.     

Semiautomatic morphometric analysis of the nucleolar development in the nucl. n. oculomotorii of Tupaia belangeri during ontogenesis

Summary The development during ontogenesis of the nuclear and nucleolar diameters and of the numbers of nucleoli per nucleus in the nucl. n. oculomotorii of 32 male Tupaia belangeri are described. The measurements of the nucleolar diameters were done automatically using the Micro-Videomat (Zeiss, Oberkochen). Increase of the nuclear and nucleolar diameters yielded a nearly sigmoid-shaped curve, which was fitted using the logistic growth function. The ideal value of the nucleolar diameter is 3.7 m with a half value time of 22 days of ontogenesis and an enlargement factor of 1.53. The respective parameters for the nucleus are 12.2 m, 28 days and 1.27. The ratio of the nuclear to nucleolar diameters decreases exponentially during ontogenesis to a value of 3.3. The validity of these easily determinable morphologic parameters for the RNA-synthesis is discussed. The number of nuclei with 2 or 3 nucleoli decreases during ontogenesis. The relevance of this fact for cell counting is also discussed.Abteilungsvorsteher: Professor Dr. H.-J. KretschmannDirektor: Professor Dr. F. WingertSupported by Deutsche Forschungsgemeinschaft.     

Characterization of the contribution of spliced RNAs of hepatitis B virus to DNA synthesis in transfected cultures of Huh7 and HepG2 cells

Hepatitis B virus synthesizes multiple spliced RNAs that can be reverse transcribed into viral DNA. We thoroughly characterized the contribution of spliced RNAs to DNA synthesis in transfected cultures of Huh7 and HepG2 cells. We found that up to 50% of DNA within intracellular capsids is derived from five spliced RNAs. Expressing HBV P protein and pgRNA from separate plasmids and the use of the CMV-IE promoter contributes to these high levels of encapsidated DNA derived from spliced RNA. A spliced RNA called Sp1 was the predominant species expressed in both cell lines. All spliced RNAs support the synthesis minus-strand DNA and duplex linear DNA. Only one of the spliced RNAs, Sp14, supported the synthesis of relaxed circular DNA because splicing removed an important cis-acting sequence (hM) in the other four RNAs. Additionally, we created a variant that was deficient in the synthesis of spliced RNA and supported DNA synthesis at wild-type levels. Our results reinforce and extend the idea that a significant fraction of HBV DNA synthesized under common experimental conditions is derived from spliced RNA. It is important that their presence be considered when analyzing HBV DNA replication in transfected cell cultures.     

儿童非何杰金淋巴瘤的DNA含量和免疫表型的研究

应用流式细胞术和免疫组化技术，对６３例儿童非何杰金淋巴瘤的ＤＮＡ含量、免疫表型和核仁组织区相关蛋白进行了研究，结果表明：异倍体率、Ａｇ－ＮＯＲ值及增殖指数（ＰＩ）随着组织学分级增高而增大，且ＰＩ可作为判断预后的一项定量指标；Ａｇ－ＮＯＲ与ＰＩ呈正相关；异倍体与二倍体肿瘤累积生存率无显著差异；累积生存率未定型淋巴瘤＞Ｔ细胞淋巴瘤＞Ｂ细胞淋巴瘤。     

Mesenchymal transition and increased therapy resistance of glioblastoma cells is related to astrocyte reactivity

Grade IV astrocytoma/glioblastoma multiforme (GBM) is essentially incurable, partly due to its heterogenous nature, demonstrated even within the glioma-initiating cell (GIC) population. Increased therapy resistance of GICs is coupled to transition into a mesenchymal (MES) cell state. The GBM MES molecular signature displays a pronounced inflammatory character and its expression vary within and between tumors. Herein, we investigate how MES transition of GBM cells relates to inflammatory responses of normal astroglia. In response to CNS insults astrocytes enter a reactive cell state and participate in directing neuroinflammation and subsequent healing processes. We found that the MES signature show strong resemblance to gene programs induced in reactive astrocytes. Likewise, astrocyte reactivity gene signatures were enriched in therapy-resistant MES-like GIC clones. Variable expression of astrocyte reactivity related genes also largely defined intratumoral GBM cell heterogeneity at the single-cell level and strongly correlated with our previously defined therapy-resistance signature (based on linked molecular and functional characterization of GIC clones). In line with this, therapy-resistant MES-like GIC secreted immunoregulatory and tissue repair related proteins characteristic of astrocyte reactivity. Moreover, sensitive GIC clones could be made reactive through long-term exposure to the proinflammatory cytokine interleukin 1 beta (IL1β). IL1β induced a slow MES transition, increased therapy resistance, and a shift in DNA methylation profile towards that of resistant clones, which confirmed a slow reprogramming process. In summary, GICs enter through MES transition a reactive-astrocyte-like cell state, connected to therapy resistance. Thus, from a biological point of view, MES GICs would preferably be called ‘reactive GICs’. The ability of GBM cells to mimic astroglial reactivity contextualizes the immunomodulatory and microenvironment reshaping abilities of GBM cells that generate a tumor-promoting milieu. This insight will be important to guide the development of future sensitizing therapies targeting treatment-resistant relapse-driving cell populations as well as enhancing the efficiency of immunotherapies in GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunolocalization of Picornavirus RNA in infected cells with antibodies to Tyr-pUp, the covalent linkage unit between VPg and RNA

The genomic RNA of picornaviruses is attached to a small protein (VPg) via a covalent bond between a tyrosine and a 5′-terminal uridine phosphate. The same structure is present in potyvirus and calicivirus families. VPgs play a key role in initiation of viral replication by acting as primers for RNA synthesis. The model compound [N(Ac),CO(NHMe)]Tyr-(5′P → O)Up-O-(CH₂)₆NH₂ (mCLU), mimicking this ‘covalent linkage unit’ (CLU) and containing Tyr-pUp was synthesized in solution following the phosphoramidite scheme and used to raise antibodies for studying picornavirus infection. The antibodies recognized CLU-containing mengovirus RNA and showed minimal cross-reactivity with RNAs lacking CLU. Immunofluorescence staining of cells infected with a human rhinovirus demonstrated co-localization of the signals from anti-mCLU and from anti-VPg antibodies. Efficient synthesis of mCLU and anti-mCLU antibodies might be of great utility for investigating viral replication and identifying yet unknown viral and cellular CLU-containing RNA-protein complexes.     

Ovarian carcinoma cells in effusions show increased S-phase fraction compared to corresponding primary tumors

The objective of this study was to analyze large-scale genomic patterns during disease progression from primary tumor to effusion in ovarian carcinoma, and to study the association between DNA ploidy parameters in effusions, proliferation/survival markers, and clinicopathologic characteristics. DNA ploidy status, DNA index (DI), and S-phase fraction (SPF) were compared in 22 matched primary carcinomas (all prechemotherapy specimens) and effusions (14 prechemotherapy and 8 postchemotherapy specimens) using image analysis. The association between these parameters and previously studied cell survival/proliferation biomarkers, previous administration of chemotherapy, chemotherapy response and survival was analyzed in a larger series of 54 effusions. The majority of specimens were aneuploid irrespective of anatomic site, with no significant differences in DI. SPF was significantly higher in effusions compared to matched primary tumors (P = 0.007 for all 22 pairs, P = 0.011 for 14 matched prechemotherapy specimens). Higher SPF was related to higher Ki-67 score (P = 0.045), and both SPF and DI were directly associated with higher level of Survivin (P < 0.001 for both). DI and SPF in effusions showed no association with histological grade, FIGO stage, residual disease volume, previous chemotherapy, response to chemotherapy at primary disease, recurrence or survival. Ovarian carcinoma cells in effusions have increased proliferation compared to corresponding primary tumors, as evidence of disease progression. DNA ploidy parameters in cancer cells in effusions are unaltered by chemotherapy and appear to be unrelated to chemotherapy response and to survival, suggesting that large-scale genomic patterns at this anatomic site are not useful in segregating patients into prognostic groups.     

Crossed resistance of tumor cells with high resistance to colchicine

Laboratory of Genetics of Tumor Cells, All-Union Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by, Academician of the Academy of Medical Sciences of the USSR N. I. Trapeznikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 108, No. 10, pp. 490–492, October, 1989.     

D3细胞的制备及其对几种病原体的敏感性研究

目的制备基因转染（D3）细胞并测定能引起先天性感染的几种病原体的敏感性.方法HEP-2细胞的DNA转染人胚肺细胞（HEL）,得到基因转染细胞,命名为D3细胞.分析D3细胞的核型.研究丹参对D3细胞促增殖作用和丹参对病原体不同的促增殖作用.普通显微镜观察病原体感染D3细胞后的细胞病变.免疫荧光试验研究D3细胞对病原体的敏感性.电子显微镜观察由D3细胞培养物纯化的病原体形态.在酶联免疫吸附试验（ELISA）中,分别纯化病原体制作抗原,检测1196份孕妇血清特异性抗体（IgG、IgM）.结果传代5～100代的D3细胞的染色体数均是96.丹参（SMB,100μg/ml）对D3细胞和病原体有明显的促增殖作用.D3细胞感染病原体后72 h,均出现明显的细胞病变效应（CPE）和荧光阳性细胞.电镜下见到了病原体典型的形态.结论永生性的D3细胞可用于培养病原体,所培养的病原体可制备用于诊断的抗原.