Introduction of a strong temperature-sensitive phenotype into enterovirus 71 by altering an amino acid of virus 3D polymerase

AbstractIn 1998, an enterovirus 71 (EV71) epidemic in Taiwan resulted in 78 deaths; however, the molecular basis of EV71 pathogenicity remains poorly understood. Comparison of the deduced amino acid sequences in 3D polymerases of EV71clinical isolates showed the T251V or T251I substitution from 1986 and 1998 outbreaks. An EV71 replicon system showed that introducing an I251T mutation did not affect luciferase activities at 35 °C when compared with wild type; however, lower luciferase activities were observed when they were incubated at 39.5 °C. In addition, the I251T mutation in the EV71 infectious clone not only reduced viral replication at 39.5 °C in vitro but also decreased the virulence of the mouse adaptive strain MP4 in neonatal mice in an i.p. infection model. Therefore, these results suggested that the threonine at position 251 results in a temperature sensitivity phenotype of EV71 which may contribute to the attenuation of circulating strains

Molecular landscape of IDH-mutant primary astrocytoma Grade IV/glioblastomas

WHO 2016 classified glioblastomas into IDH-mutant and IDH-wildtype with the former having a better prognosis but there was no study on IDH-mutant primary glioblastomas only, as previous series included secondary glioblastomas. We recruited a series of 67 IDH-mutant primary glioblastomas/astrocytoma IV without a prior low-grade astrocytoma and examined them using DNA-methylation profiling, targeted sequencing, RNA sequencing and TERT promoter sequencing, and correlated the molecular findings with clinical parameters. The median OS of 39.4 months of 64 cases and PFS of 25.9 months of 57 cases were better than the survival data of IDH-wildtype glioblastomas and IDH-mutant secondary glioblastomas retrieved from datasets. The molecular features often seen in glioblastomas, such as EGFR amplification, combined +7/-10, and TERT promoter mutations were only observed in 6/53 (11.3%), 4/53 (7.5%), and 2/67 (3.0%) cases, respectively, and gene fusions were found only in two cases. The main mechanism for telomere maintenance appeared to be alternative lengthening of telomeres as ATRX mutation was found in 34/53 (64.2%) cases. In t-SNE analyses of DNA-methylation profiles, with an exceptional of one case, a majority of our cases clustered to IDH-mutant high-grade astrocytoma subclass (40/53; 75.5%) and the rest to IDH-mutant astrocytoma subclass (12/53; 22.6%). The latter was also enriched with G-CIMP high cases (12/12; 100%). G-CIMP-high status and MGMT promoter methylation were independent good prognosticators for OS (p = 0.022 and p = 0.002, respectively) and TP53 mutation was an independent poor prognosticator (p = 0.013) when correlated with other clinical parameters. Homozygous deletion of CDKN2A/B was not correlated with OS (p = 0.197) and PFS (p = 0.278). PDGFRA amplification or mutation was found in 16/59 (27.1%) of cases and was correlated with G-CIMP-low status (p = 0.010). Aside from the three well-known pathways of pathogenesis in glioblastomas, chromatin modifying and mismatch repair pathways were common aberrations (88.7% and 20.8%, respectively), the former due to high frequency of ATRX involvement. We conclude that IDH-mutant primary glioblastomas have better prognosis than secondary glioblastomas and have major molecular differences from other commoner glioblastomas. G-CIMP subgroups, MGMT promoter methylation, and TP53 mutation are useful prognostic adjuncts

A Single Nucleotide in Stem Loop II of 5′-Untranslated Region Contributes to Virulence of Enterovirus 71 in Mice

BACKGROUND: Enterovirus 71 (EV71) has emerged as a neuroinvasive virus responsible for several large outbreaks in the Asia-Pacific region while virulence determinant remains unexplored. PRINCIPAL FINDINGS: In this report, we investigated increased virulence of unadapted EV71 clinical isolate 237 as compared with isolate 4643 in mice. A fragment 12 nucleotides in length in stem loop (SL) II of 237 5'-untranslated region (UTR) visibly reduced survival time and rate in mice was identified by constructing a series of infectious clones harboring chimeric 5'-UTR. In cells transfected with bicistronic plasmids, and replicon RNAs, the 12-nt fragment of isolate 237 enhanced translational activities and accelerated replication of subgenomic EV71. Finally, single nucleotide change from cytosine to uridine at base 158 in this short fragment of 5'-UTR was proven to reduce viral translation and EV71 virulence in mice. Results collectively indicated a pivotal role of novel virulence determinant C158 on virus translation in vitro and EV71 virulence in vivo. CONCLUSIONS: These results presented the first reported virulence determinant in EV71 5'-UTR and first position discovered from unadapted isolates

A new prognostic histopathologic classification of nasopharyngeal carcinoma

Background: The current World Health Organization (WHO) classification of nasopharyngeal carcinoma (NPC) conveys little prognostic information. This study aimed to propose an NPC histopathologic classification that can potentially be used to predict prognosis and treatment response. Methods: We initially developed a histopathologic classification based on the morphologic traits and cell differentiation of tumors of 2716 NPC patients who were identified at Sun Yat-sen University Cancer Center (SYSUCC) (training cohort). Then, the proposed classification was applied to 1702 patients (retrospective validation cohort) from hospitals outside SYSUCC and 1613 patients (prospective validation cohort) from SYSUCC. The efficacy of radiochemotherapy and radiotherapy modalities was compared between the proposed subtypes. We used Cox proportional hazards models to estimate hazard ratios (HRs) with 95% confidence intervals (CI) for overall survival (OS). Results: The 5-year OS rates for all NPC patients who were diagnosed with epithelial carcinoma (EC; 3708 patients), mixed sarcomatoid-epithelial carcinoma (MSEC; 1247 patients), sarcomatoid carcinoma (SC; 823 patients), and squamous cell carcinoma (SCC; 253 patients) were 79.4%, 70.5%, 59.6%, and 42.6%, respectively (P < 0.001). In multivariate models, patients with MSEC had a shorter OS than patients with EC (HR = 1.44, 95% CI = 1.27–1.62), SC (HR = 2.00, 95% CI = 1.76–2.28), or SCC (HR = 4.23, 95% CI = 3.34–5.38). Radiochemotherapy significantly improved survival compared with radiotherapy alone for patients with EC (HR = 0.67, 95% CI = 0.56–0.80), MSEC (HR = 0.58, 95% CI = 0.49–0.75), and possibly for those with SCC (HR = 0.63; 95% CI = 0.40–0.98), but not for patients with SC (HR = 0.97, 95% CI = 0.74–1.28). Conclusions: The proposed classification offers more information for the prediction of NPC prognosis compared with the WHO classification and might be a valuable tool to guide treatment decisions for subtypes that are associated with a poor prognosis

Subneutralizing antibodies to enterovirus 71 induce antibody-dependent enhancement of infection in newborn mice

Antibody-dependent enhancement (ADE) of virus infections can be induced by subneutralizing concentrations of specific antibodies. We recently demonstrated ADE in human monocytes infected with enterovirus 71 (EV71). The current study was designed to extend these observations by determining the effect of ADE on the pathogenesis of EV71 infection in newborn mice. We compared the clinical manifestations, mortality, virus titer, histopathology, and serum levels of cytokines and chemokines in newborn mice pretreated with subneutralizing antibodies to EV71 or normal mouse IgG with and without virus. Seven-day-old ICR mice were pretreated with a wide range of mouse anti-EV71 IgG 24 h prior to intraperitoneal injection of EV71. Mice were protected from infection by neutralizing doses of anti-EV71 IgG ranging from 6.43 × 10-1 to 329.6 μg/ml. Subneutralizing doses ranging from 2.01 × 10-2 to 3.21 × 10-1 μg/ml were found to significantly increase 14-day mortality compared to virus alone. The ADE effect was not evident at lower doses. Histopathological examination of mice given a subneutralizing dose of 8.04 × 10-2 μg/ml revealed extensive neuronal and muscular damage compared to untreated infected controls. Higher serum levels of interferon (IFN)-γ and monocyte chemoattractant protein (MCP)-1 were noted in mice pretreated with subneutralizing doses than untreated infected controls. These findings support the concept that subneutralizing antibodies directed enhance EV71 induce ADE in newborn mice. © 2013 Springer-Verlag Berlin Heidelberg

APLNR marks a cardiac progenitor derived with human induced pluripotent stem cells

Cardiomyocytes can be readily derived from human induced pluripotent stem cell (hiPSC) lines, yet its efficacy varies across different batches of the same and different hiPSC lines. To unravel the inconsistencies of in vitro cardiac differentiation, we utilized single cell transcriptomics on hiPSCs undergoing cardiac differentiation and identified cardiac and extra-cardiac lineages throughout differentiation. We further identified APLNR as a surface marker for in vitro cardiac progenitors and immunomagnetically isolated them. Differentiation of isolated in vitro APLNR+ cardiac progenitors derived from multiple hiPSC lines resulted in predominantly cardiomyocytes accompanied with cardiac mesenchyme. Transcriptomic analysis of differentiating in vitro APLNR+ cardiac progenitors revealed transient expression of cardiac progenitor markers before further commitment into cardiomyocyte and cardiac mesenchyme. Analysis of in vivo human and mouse embryo single cell transcriptomic datasets have identified APLNR expression in early cardiac progenitors of multiple lineages. This platform enables generation of in vitro cardiac progenitors from multiple hiPSC lines without genetic manipulation, which has potential applications in studying cardiac development, disease modelling and cardiac regeneration

Norepinephrine and Epinephrine Enhanced the Infectivity of Enterovirus 71

<div><p>Background</p><p>Enterovirus 71 (EV71) infections may be associated with neurological complications, including brainstem encephalitis (BE). Severe EV71 BE may be complicated with autonomic nervous system (ANS) dysregulation and/or pulmonary edema (PE). ANS dysregulation is related to the overactivation of the sympathetic nervous system, which results from catecholamine release.</p><p>Objective</p><p>The aims of this study were to explore the effects of catecholamines on severe EV71 infection and to investigate the changes in the percentages of EV71-infected cells, virus titer, and cytokine production on the involvement of catecholamines.</p><p>Study Design</p><p>Plasma levels of norepinephrine (NE) and epinephrine (EP) in EV71-infected patients were measured using an enzyme-linked immunoassay. The expression of adrenergic receptors (ADRs) on RD, A549, SK-N-SH, THP-1, Jurkat and human peripheral blood mononuclear cells (hPBMCs) were detected using flow cytometry. The percentages of EV71-infected cells, virus titer, and cytokine production were investigated after treatment with NE and EP.</p><p>Results</p><p>The plasma levels of NE and EP were significantly higher in EV71-infected patients with ANS dysregulation and PE than in controls. Both α_1A- and β₂-ADRs were expressed on A549, RD, SK-N-SH, HL-60, THP-1, Jurkat cells and hPBMCs. NE treatment elevated the percentages of EV71-infected cells to 62.9% and 22.7% in THP-1 and Jurkat cells, respectively. Via treatment with EP, the percentages of EV71-infected cells were increased to 64.6% and 26.9% in THP-1 and Jurkat cells. The percentage of EV71-infected cells increased upon NE or EP treatment while the α- and β-blockers reduced the percentages of EV71-infected cells with NE or EP treatment. At least two-fold increase in virus titer was observed in EV71-infected A549, SK-N-SH and hPBMCs after treatment with NE or EP. IL-6 production was enhanced in EV71-infected hPBMCs at a concentration of 10² pg/mL NE.</p><p>Conclusion</p><p>The plasma levels of NE and EP elevated in EV71-infected patients with ANS dysregulation and PE. Both NE and EP enhanced the percentages of infected cells and virus titers in EV71 infection <i>in vitro</i>. NE and EP may play a role in the pathogenesis of EV71 BE complicated with ANS dysregulation and PE.</p></div