The Amino-Terminal Domain of Alphavirus Capsid Protein Is Dispensable for Viral Particle Assembly but Regulates RNA Encapsidation through Cooperative Functions of Its Subdomains

Venezuelan equine encephalitis virus (VEEV) is a pathogenic alphavirus, which circulates in the Central, South, and North Americas, including the United States, and represents a significant public health threat. In recent years, strong progress has been made in understanding the structure of VEEV virions, but the mechanism of their formation has yet to be investigated. In this study, we analyzed the functions of different capsid-specific domains and its amino-terminal subdomains in viral particle formation. Our data demonstrate that VEEV particles can be efficiently formed directly at the plasma membrane without cytoplasmic nucleocapsid preassembly. The entire amino-terminal domain of VEEV capsid protein was found to be dispensable for particle formation. VEEV variants encoding only the capsid''s protease domain efficiently produce genome-free VEEV virus-like particles (VLPs), which are very similar in structure to the wild-type virions. The amino-terminal domain of the VEEV capsid protein contains at least four structurally and functionally distinct subdomains, which mediate RNA packaging and the specificity of packaging in particular. The most positively charged subdomain is a negative regulator of the nucleocapsid assembly. The three other subdomains are not required for genome-free VLP formation but are important regulators of RNA packaging. Our data suggest that the positively charged surface of the VEEV capsid-specific protease domain and the very amino-terminal subdomain are also involved in interaction with viral RNA and play important roles in RNA encapsidation. Finally, we show that VEEV variants with mutated capsid acquire compensatory mutations in either capsid or nsP2 genes.     

Protein kinase D protects against oxidative stress-induced intestinal epithelial cell injury via Rho/ROK/PKC-delta pathway activation

Protein kinase D (PKD) is a novel protein serine kinase that has recently been implicated in diverse cellular functions, including apoptosis and cell proliferation. The purpose of our present study was 1) to define the activation of PKD in intestinal epithelial cells treated with H₂O₂, an agent that induces oxidative stress, and 2) to delineate the upstream signaling mechanisms mediating the activation of PKD. We found that the activation of PKD is induced by H₂O₂ in both a dose- and time-dependent fashion. PKD phosphorylation was attenuated by rottlerin, a selective PKC- inhibitor, and by small interfering RNA (siRNA) directed against PKC-, suggesting the regulation of PKD activity by upstream PKC-. Activation of PKD was also blocked by a Rho kinase (ROK)-specific inhibitor, Y-27632, as well as by C3, a Rho protein inhibitor, demonstrating that the Rho/ROK pathway also mediates PKD activity in intestinal cells. In addition, H₂O₂-induced PKC- phosphorylation was inhibited by C3 treatment, further suggesting that PKC- is downstream of Rho/ROK. Interestingly, H₂O₂-induced intestinal cell apoptosis was enhanced by PKD siRNA. Together, these results clearly demonstrate that oxidative stress induces PKD activation in intestinal epithelial cells and that this activation is regulated by upstream PKC- and Rho/ROK pathways. Importantly, our findings suggest that PKD activation protects intestinal epithelial cells from oxidative stress-induced apoptosis. These findings have potential clinical implications for intestinal injury associated with oxidative stress (e.g., necrotizing enterocolitis in infants). Rho kinase; protein kinase C-     

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules

Background

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.

Results

By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca²⁺ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.

Conclusions

Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.     

Identification of microRNAs as potential prognostic markers in ependymoma

Introduction

We have examined expression of microRNAs (miRNAs) in ependymomas to identify molecular markers of value for clinical management. miRNAs are non-coding RNAs that can block mRNA translation and affect mRNA stability. Changes in the expression of miRNAs have been correlated with many human cancers.

Materials and Methods

We have utilized TaqMan Low Density Arrays to evaluate the expression of 365 miRNAs in ependymomas and normal brain tissue. We first demonstrated the similarity of expression profiles of paired frozen tissue (FT) and paraffin-embedded specimens (FFPE). We compared the miRNA expression profiles of 34 FFPE ependymoma samples with 8 microdissected normal brain tissue specimens enriched for ependymal cells. miRNA expression profiles were then correlated with tumor location, histology and other clinicopathological features.

Results

We have identified miRNAs that are over-expressed in ependymomas, such as miR-135a and miR-17-5p, and down-regulated, such as miR-383 and miR-485-5p. We have also uncovered associations between expression of specific miRNAs which portend a worse prognosis. For example, we have identified a cluster of miRNAs on human chromosome 14q32 that is associated with time to relapse. We also found that miR-203 is an independent marker for relapse compared to the parameters that are currently used. Additionally, we have identified three miRNAs (let-7d, miR-596 and miR-367) that strongly correlate to overall survival.

Conclusion

We have identified miRNAs that are differentially expressed in ependymomas compared with normal ependymal tissue. We have also uncovered significant associations of miRNAs with clinical behavior. This is the first report of clinically relevant miRNAs in ependymomas.     

In vitro assessment of some sperm function following exposure to levonorgestrel in human fallopian tubes

Background  

The mechanism of action of levonorgestrel (LNG) as emergency contraception (EC) remains a subject of debate and its effect on sperm function has been only partially explained. The aim of this study was to assess whether LNG at a similar dose to those found in serum following oral intake for EC could affect spermatozoa when exposed to human fallopian tubes in vitro.     

Single agent and synergistic combinatorial efficacy of first-in-class small molecule imipridone ONC201 in hematological malignancies

ONC201, founding member of the imipridone class of small molecules, is currently being evaluated in advancer cancer clinical trials. We explored single agent and combinatorial efficacy of ONC201 in preclinical models of hematological malignancies. ONC201 demonstrated (GI50 1–8 µM) dose- and time-dependent efficacy in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), Burkitt's lymphoma, anaplastic large cell lymphoma (ALCL), cutaneous T-cell lymphoma (CTCL), Hodgkin's lymphoma (nodular sclerosis) and multiple myeloma (MM) cell lines including cells resistant to standard of care (dexamethasone in MM) and primary samples. ONC201 induced caspase-dependent apoptosis that involved activation of the integrated stress response (ATF4/CHOP) pathway, inhibition of Akt phosphorylation, Foxo3a activation, downregulation of cyclin D1, IAP and Bcl-2 family members. ONC201 synergistically reduced cell viability in combination with cytarabine and 5-azacytidine in AML cells. ONC201 combined with cytarabine in a Burkitt's lymphoma xenograft model induced tumor growth inhibition that was superior to either agent alone. ONC201 synergistically combined with bortezomib in MM, MCL and ALCL cells and with ixazomib or dexamethasone in MM cells. ONC201 combined with bortezomib in a Burkitt's lymphoma xenograft model reduced tumor cell density and improved CHOP induction compared to either agent alone. These results serve as a rationale for ONC201 single-agent trials in relapsed/refractory acute leukemia, non-Hodgkin's lymphoma, MM and combination trial with dexamethasone in MM, provide pharmacodynamic biomarkers and identify further synergistic combinatorial regimens that can be explored in the clinic.     

Characterization of calcium-dependent membrane binding proteins of brain cortex.

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr-32 000 and -34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.