Betanodavirus Induces Oxidative Stress-Mediated Cell Death That Prevented by Anti-Oxidants and Zfcatalase in Fish Cells

The role of oxidative stress in the pathogenesis of RNA nervous necrosis virus infection is still unknown. Red-spotted grouper nervous necrosis virus (RGNNV) induced free radical species (ROS) production at 12–24 h post-infection (pi; early replication stage) in fish GF-1 cells, and then at middle replication stage (24–48 h pi), this ROS signal may upregulate some expressions of the anti-oxidant enzymes Cu/Zn SOD and catalase, and eventually expression of the transcription factor Nrf2. Furthermore, both antioxidants diphenyliodonium and N-acetylcysteine or overexpression of zebrafish catalase in GF-1 cells also reduced ROS production and protected cells for enhancing host survival rate due to RGNNV infection

Mutation and Lineage Analysis of DNMT3A in BCR-ABL1-negative Chronic Myeloproliferative Neoplasms

SummaryIn addition to the JAK2 V617F mutation, somatic mutation in DNMT3A has been described in BCL-ABL1-negative myeloproliferative neoplasms (MPNs). We have screened for DNMT3A exon 23 mutations in 130 adult Taiwanese patients with chronic phase myeloproliferative neoplasms. Only one somatic DNMT3A R882H mutation was identified in one JAK2 V617F mutation-positive essential thrombocythemia patient (1/91, 1%). Both mutations were detected in the CD34+-, CD19+-, peripheral blood mononuclear cell- and granulocyte-enriched fractions, but were not detected in the CD3+-enriched fraction by lineage analysis. Our findings suggest that DNMT3A mutation is not prevalent in MPNs, and further study is needed to clarify its role in the molecular pathogenesis of myeloproliferative neoplasms

Association of HLA class I with severe acute respiratory syndrome coronavirus infection

BACKGROUND: The human leukocyte antigen (HLA) system is widely used as a strategy in the search for the etiology of infectious diseases and autoimmune disorders. During the Taiwan epidemic of severe acute respiratory syndrome (SARS), many health care workers were infected. In an effort to establish a screening program for high risk personal, the distribution of HLA class I and II alleles in case and control groups was examined for the presence of an association to a genetic susceptibly or resistance to SARS coronavirus infection. METHODS: HLA-class I and II allele typing by PCR-SSOP was performed on 37 cases of probable SARS, 28 fever patients excluded later as probable SARS, and 101 non-infected health care workers who were exposed or possibly exposed to SARS coronavirus. An additional control set of 190 normal healthy unrelated Taiwanese was also used in the analysis. RESULTS: Woolf and Haldane Odds ratio (OR) and corrected P-value (Pc) obtained from two tails Fisher exact test were used to show susceptibility of HLA class I or class II alleles with coronavirus infection. At first, when analyzing infected SARS patients and high risk health care workers groups, HLA-B*4601 (OR = 2.08, P = 0.04, Pc = n.s.) and HLA-B*5401 (OR = 5.44, P = 0.02, Pc = n.s.) appeared as the most probable elements that may be favoring SARS coronavirus infection. After selecting only a "severe cases" patient group from the infected "probable SARS" patient group and comparing them with the high risk health care workers group, the severity of SARS was shown to be significantly associated with HLA-B*4601 (P = 0.0008 or Pc = 0.0279). CONCLUSIONS: Densely populated regions with genetically related southern Asian populations appear to be more affected by the spreading of SARS infection. Up until recently, no probable SARS patients were reported among Taiwan indigenous peoples who are genetically distinct from the Taiwanese general population, have no HLA-B* 4601 and have high frequency of HLA-B* 1301. While increase of HLA-B* 4601 allele frequency was observed in the "Probable SARS infected" patient group, a further significant increase of the allele was seen in the "Severe cases" patient group. These results appeared to indicate association of HLA-B* 4601 with the severity of SARS infection in Asian populations. Independent studies are needed to test these results

GABB : A global dataset of alpine breeding birds and their ecological traits

Alpine ecosystems represent varied climates and vegetation structures globally, with the potential to support rich and functionally diverse avian communities. High mountain habitats and species are under significant threat from climate change and other anthropogenic factors. Yet, no global database of alpine birds exists, with most mountain systems lacking basic information on species breeding in alpine habitats, their status and trends, or potential cryptic diversity (i.e., sub-species distributions). To address these critical knowledge gaps, we combined published literature, regional monitoring schemes, and expert knowledge from often inaccessible, data-deficient mountain ranges to develop a global list of alpine breeding bird species with their associated distributions and select ecological traits. This dataset compiles alpine breeding records for 1,310 birds, representing 12.0% of extant species and covering all major mountain regions across each continent, excluding Antarctica. The Global Alpine Breeding Bird dataset (GABB) is an essential resource for research on the ecological and evolutionary factors shaping alpine communities, as well as documenting the value of these high elevation, climate-sensitive habitats for conserving biodiversity.Peer reviewe

NF-κB is activated in CD4+ iNKT cells by sickle cell disease and mediates rapid induction of adenosine A2A receptors.

Reperfusion injury following tissue ischemia occurs as a consequence of vaso-occlusion that is initiated by activation of invariant natural killer T (iNKT) cells. Sickle cell disease (SDC) results in widely disseminated microvascular ischemia and reperfusion injury as a result of vaso-occlusion by rigid and adhesive sickle red blood cells. In mice, iNKT cell activation requires NF-κB signaling and can be inhibited by the activation of anti-inflammatory adenosine A2A receptors (A2ARs). Human iNKT cells are divided into subsets of CD4+ and CD4- cells. In this study we found that human CD4+ iNKT cells, but not CD4- cells undergo rapid NF-κB activation (phosphorylation of NF-κB on p65) and induction of A2ARs (detected with a monoclonal antibody 7F6-G5-A2) during SCD painful vaso-occlusive crises. These findings indicate that SCD primarily activates the CD4+ subset of iNKT cells. Activation of NF-κB and induction of A2ARs is concordant, i.e. only CD4+ iNKT cells with activated NF-κB expressed high levels of A2ARs. iNKT cells that are not activated during pVOC express low levels of A2AR immunoreactivity. These finding suggest that A2AR transcription may be induced in CD4+ iNKT cells as a result of NF-κB activation in SCD. In order to test this hypothesis further we examined cultured human iNKT cells. In cultured cells, blockade of NF-κB with Bay 11-7082 or IKK inhibitor VII prevented rapid induction of A2AR mRNA and protein upon iNKT activation. In conclusion, NF-κB-mediated induction of A2ARs in iNKT cells may serve as a counter-regulatory mechanism to limit the extent and duration of inflammatory immune responses. As activated iNKT cells express high levels of A2ARs following their activation, they may become highly sensitive to inhibition by A2AR agonists

Identification of RGNNV infection induces ROS production in GF-1 cells.

<p>(A) ROS production (indicated by arrows) at 0, 12, 24, 48, and 72 h pi by cells infected with RGNNV (MOI = 1). The negative control (0 h) (see e and i); 24 h negative control (see a and b); RGNNV-infected groups at 12, 24, 48, and 72 h pi (see f–m and j–n, respectively; Green fluorescent cells as the ROS production positive cells); positive control H₂O₂ (1 µM) at 24 h post-incubation (see c and d; Green fluorescent cells as the ROS production positive cells). Scale bar = 10 µm. (B) The percentage of ROS-producing cells was counted at 0, 12, 24, 48, and 72 h, and shows a significant increase over time. In this and all subsequent figures (unless otherwise noted) data are presented as the percentage of 200 cells at each time point determined in triplicate, with each point representing the mean of three independent experiments. The vertical bars indicate ± the standard error of the mean (SEM). All data were analyzed using either a paired or unpaired Student's <i>t</i>-test, as appropriate. Statistically significant was defined at <i>P</i><0.01. (C) The ratios of ROS-producing cells were counted by fluorescence microplate reader at 0, 12, 24, 48, and 72 h, and showed a significant increase at 24 h pi, but not at 48 h and 72 h pi because those times left few cells in wells. *<i>P</i><0.01 indicated a statistically significant difference between mean values of the groups. (D) Concentration of peroxide in medium of RGNNV-infected cells producing H₂O₂ ratio at 12, 24, 48, and 72 h pi, respectively. Peroxide concentration at each time point was determined in triplicate. *<i>P</i><0.05.</p

Identification of zebrafish catalase overexpression can reduce RGNNV-induced ROS-mediated cell death and viral titers in GF-1 cells.

<p>(A) Western blot analysis of zebrafish catalase-producing cell lines in GF-1 cells after selection with Zeocin (500 µg/ml). The stable clones are zfCatalase-1 (lane 2), zfCatalase-3 (lane 3), and vector control-4 (lane 1). HeLa cell lysate serves as a positive control (lane 4). Actin used as an internal loading control. (B) The number of ROS-producing cells after infection with RGNNV (MOI = 1) at 0, 48, and 72 h. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.01. (C) The viability of cells transfected with vector control-4 or zfcatalase-3 and infected with RGNNV was determined at 0, 48, and 72 h pi in triplicate by using a trypan blue dye exclusion assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025853#pone.0025853-Mullen1" target="_blank">[37]</a>. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.01. (D) Viral titers were assays in GF-1 cell line by using at 48 h and 72 h pi samples. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.05.</p

Influence of anti-oxidants NAC and DPI treatments on ROS production and cellular viability during RGNNV infection.

<p>(A) The GF-1 cells pre-treated with antioxidants either NAC or DPI for two hours, then infected with RGNNV (MOI = 1) for different time incubations. The number of ROS producing cells infected with RGNNV TN1 at 0, 24, 48, and 72 h pi was assayed with the Image-iT LIVE Green Reactive Oxygen Species Detection Kit. Data are the percentage of 200 cells at each time point, determined in triplicate, with each point representing the mean of three independent experiments; error bars represent the SEM. The data were analyzed using either a paired or unpaired Student's <i>t</i>-test. as appropriate. *<i>P</i><0.01. (B) The viability of GF-1 cells infected with RGNNV and treated with or without NAC or DPI at 0, 24, 48, and 72 h pi in triplicate by using a trypan blue dye exclusion assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025853#pone.0025853-Mullen1" target="_blank">[37]</a>. The data were analyzed using either a paired or unpaired Student's <i>t</i>-test. as appropriate. *<i>P</i><0.05.</p

Western blot analysis of RGNNV infection up-regulates anti-oxidant enzymes Cu/Zn SOD and catalase or transcriptional factor Nrf2 in GF-1 cells at middle replication stage.

<p>The GF-1 cells pre-treated with antioxidants either NAC (1 mM) or DPI (30 mM) for two hours, then infected with RGNNV (MOI = 1) for different time incubations at 0, 24, 48, and 72 h pi. Samples were electrophoresed on a SDS-polyacrylamine gel and electro-blotted to a NC membrane. The NC membrane was stained with mouse monoclonal IgG antibodies directed against Cu/Zn SOD (15-kDa; Cayman), Catalase (57-kDa; Rockland), and Nrf2 (74-kDa) (Rockland). The chemiluminescent signal was imaged on XAR-5 film (Kodak) using a 5-min exposure. Lanes 1–4, 30 ul of virus-infected GF-1 cells and corresponded to 0, 24 h, 48 h and 72 h pi, respectively. The actin internal is also shown.</p

Identification of anti-oxidants treatment can reduce apoptotic/necrotic death of cells infected with RGNNV.

<p>(A) Phase-contrast and fluorescent micrographs of annexin-V–stained, RGNNV-infected GF-1 cells without drug-treatment at 0 h (a and f), 24 h (b and g), 48 h (c and h), and 72 h (d and i) or with NAC-treatment at 24 h (e and j), 48 h (k and p), and 72 h (l and q) and DPA-treatment at 24 h (m and r), 48 h (n and s), and 72 h (o and t). Annexin-V–positive cells (necrotic cells) are indicated by arrows. Scale bar = 20 µm. (B) The number of annexin-V–positive cells after infection with RGNNV at 0, 24, 48, and 72 h. Statistical comparisons were made using either a paired or unpaired Student's <i>t</i>-test, as appropriate. *<i>P</i><0.05. (C) Examples of flow cytometric profiles in 48 h pi. RGNNV-infected cell and plus anti-oxidants treatment cells PI staining fluorescence was measured from 10,000 cells. Numbers in second peak scales (PI⁺) show late apoptotic/secondary necrotic cell percentages respectively. Viable cell percentage (PI⁻) is shown in first peak.</p