Identification of a novel regulatory sequence of actin nucleation promoting factor encoded by Autographa californica multiple nucleopolyhedrovirus.

Actin polymerization induced by nucleation promoting factors (NPFs) is one of the most fundamental biological processes in eukaryotic cells. NPFs contain a conserved output domain (VCA domain) near the C terminus, which interacts with and activates the cellular actin-related protein 2/3 complex (Arp2/3) to induce actin polymerization and a diverse regulatory domain near the N terminus. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) nucleocapsid protein P78/83 is a virus-encoded NPF that contains a C-terminal VCA domain and induces actin polymerization in virus-infected cells. However, there is no similarity between the N terminus of P78/83 and that of other identified NPFs, suggesting that P78/83 may possess a unique regulatory mechanism. In this study, we identified a multifunctional regulatory sequence (MRS) located near the N terminus of P78/83 and determined that one of its functions is to serve as a degron to mediate P78/83 degradation in a proteasome-dependent manner. In AcMNPV-infected cells, the MRS also binds to another nucleocapsid protein, BV/ODV-C42, which stabilizes P78/83 and modulates the P78/83-Arp2/3 interaction to orchestrate actin polymerization. In addition, the MRS is also essential for the incorporation of P78/83 into the nucleocapsid, ensuring virion mobility powered by P78/83-induced actin polymerization. The triple functions of the MRS enable P78/83 to serve as an essential viral protein in the AcMNPV replication cycle, and the possible roles of the MRS in orchestrating the virus-induced actin polymerization and viral genome decapsidation are discussed.

A novel combined fluorescent probe staining method for circulating tumor cell identification.

Background: To develop a novel highly accurate circulating tumor cell (CTC) identification method and to validate its application in cancer diagnostics and/or prognostics. Methods: We verified and validated the combined fluorescent probe staining protocol (combination of three fluorescent probes: Dil, Hoechst 33342, and PY) through CTC and non-CTC (white blood cell) morphological comparison of five tumor cell lines (THP-1, HEC, HEPG2, Eca-109, HeLa) in vitro and 32 patient tumor samples from the Shandong Cancer Hospital and Institute. Wright's Giemsa staining and cluster differentiation 45 (CD45) immunocytochemistry (ICC) staining were used as reference control methods. The association between the developed method and clinicopathology was also investigated. Results: We successfully developed and optimized the protocol, and validated the use of combined fluorescent probe staining for the identification of CTCs in the peripheral blood (PB) of tumor cell lines and tumor patients. Comparable CTC and non-CTC morphologies were observed for combined fluorescent probe staining and Giemsa staining methods in vitro. However, in vivo comparison between the three staining methods revealed that the identified CTCs differed in cell diameter and nucleo-cytoplasmic ratio. In addition, a higher CTC detection rate of 14/32, lower standard deviation (SD), and higher area under the receiver operating characteristic (ROC) curve (AUC) value of 0.844 were noted for combined fluorescence staining. Clinicopathological analysis revealed that CTCs were correlated with platelet levels (P=0.031), but not with age, gender, drinking history, or granule ratio. Conclusions: We developed a combined fluorescent probe staining method with higher CTC identification accuracy than Wright's Giemsa staining, and propose this technique as a novel clinical diagnostic/prognostic tool.

Isolation and characterization of a novel alphanodavirus.

BACKGROUND: Nodaviridae is a family of non-enveloped isometric viruses with bipartite positive-sense RNA genomes. The Nodaviridae family consists of two genera: alpha- and beta-nodavirus. Alphanodaviruses usually infect insect cells. Some commercially available insect cell lines have been latently infected by Alphanodaviruses. RESULTS: A non-enveloped small virus of approximately 30 nm in diameter was discovered co-existing with a recombinant Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) in Hz-AM1 cells. Genome sequencing and phylogenetic assays indicate that this novel virus belongs to the genus of alphanodavirus in the family Nodaviridae and was designated HzNV. HzNV possesses a RNA genome that contains two segments. RNA1 is 3038 nt long and encodes a 110 kDa viral protein termed protein A. The 1404 nt long RNA2 encodes a 44 kDa protein, which exhibits a high homology with coat protein precursors of other alphanodaviruses. HzNV virions were located in the cytoplasm, in association with cytoplasmic membrane structures. The host susceptibility test demonstrated that HzNV was able to infect various cell lines ranging from insect cells to mammalian cells. However, only Hz-AM1 appeared to be fully permissive for HzNV, as the mature viral coat protein essential for HzNV particle formation was limited to Hz-AM1 cells. CONCLUSION: A novel alphanodavirus, which is 30 nm in diameter and with a limited host range, was discovered in Hz-AM1 cells.

Distribution of radon and risk assessment of its radiation dose in groundwater drinking for village people nearby the W-polymetallic metallogenic district at Dongpo in southern Hunan province, China.

Radon in the household water (especially groundwater) which is an important source of indoor radon, has become a potential health hazard to residents. In this study, radon concentrations in groundwater sampled from five villages near Dongpo W-polymetallic metallogenic region were measured using RAD-7 detector with RAD H2O accessory, and the effect of regional geology and mineralization on radon concentration in groundwater was studied. In addition, we also estimated the radiation doses received by people via ingestion of radon in water and inhalation of the radon from the indoor air while using water. The results show that the radon concentration in groundwater samples varies from 1.29 Bq L-1 to 31.31 Bq L-1 with 10.47 Bq L-1 on average, and about 31.3% of the groundwater samples analyzed have a higher radon concentration than the maximum contaminant level of 11.1 Bq L-1 recommended by United States Environmental Protection Agency (USEPA). The relatively high radon level in groundwater can be attributed to a relatively high uranium background produced by the magmatic activity and magmatic-hydrothermal system. The values of annual effective dose (AEDing) due to ingestion of radon in groundwater range from 0.002 mSv y-1 to 0.055 mSv y-1, 0.005 mSv y-1 to 0.11 mSv y-1 and 0.008 mSv y-1 to 0.188 mSv y-1 for adult, child and infant respectively. The values of annual effective dose due to the inhalation of radon released from water are 63.6, 15.4 and 3.8 times of those through the ingestion of radon in groundwater by the adults, children and infants, respectively. In addition, the values of estimated total annual effective doses are 0.020-0.480 mSv y-1, 0.017-0.406 mSv y-1 and 0.020-0.484 mSv y-1 for adult, child and infant, respectively. These values are much lower than the reference dose level of 1 mSv y-1 recommended by World Health Organization (WHO) and United Nations Scientific Committee on the Effect of Atomic Radiation (UNSCEAR).

[Cloning and functional research of Arp2/3-P40/ARPC1 subunit of Sf9 cells].

The baculovirus-induced actin polymerization is mainly associated with the virus nucleocapsid protein P78/83, which is homologous with WASP proteins that can activate Arp2/3 complex and induce the actin polymerization. In order to explore the role of Arp2/3 complex in the baculovirus replication, the P40 subunit of Arp2/3 complex from Sf9 (Spodoptera frugiperda 9) cell line was cloned and characterized. Immunofluorescent microscopy assay indicated that P40 was recruited to the inner-side of nuclear membrane during virus infection, which was in accordance with nuclear F-actin distribution in virus-infected cells as documented in our previous research, suggesting P40 could be used to track Arp2/3 complex subcellular distribution changes during virus infection. In addition, co-immunoprecipitation assay demonstrated that P40 interacted with P78/83 only in virus-infected cells, suggesting that actin polymerization induced by P78/83-Arp2/3 complex during baculovirus infection was regulated by some unidentified virus factors.