<i>In vitro</i> polymerase activities of vRNPs and plaque formation of recombinant viruses.

<p>(A) Polymerase activities of reconstituted HB04 and HN05 vRNP complexes composed of the indicated plasmids. 293T cells were transfected with the pPolI-NS-Luc plasmid and pRL-TK (internal control plasmid) as well as plasmids expressing PB2, PB1, PA and NP derived from either the HB04 or HN05 virus. Cells were incubated at 37°C for 24 h, and Firefly and Renilla luciferase activities were measured in the cell lysates. The data are represented as the means ± SD of the three independent experiments, expressed as log₁₀ relative fold to HB04 RNP activity. (B) Plaque formation after virus titration in MDCK cells.</p

Growth characterization of two H5N1 isolates.

<p>(A) Plaque phenotypes of HB04 and HN05 viruses. Plaque assays were performed using MDCK cells under standard conditions followed by staining with crystal violet. (B) Replication kinetics of the HB04 and HN05 viruses <i>in vitro</i>. Monolayers of A549, MDCK, or DF-1 cells were infected at a multiplicity of 0.1, 0.01, or 0.01 PFU/cell with HB04 and HN05, respectively. Supernatants were collected at 8, 12, 24, 48 and 72 h p.i., and viral titers were determined using MDCK cells.</p

Replication <i>in vivo</i> and pathogenicity of recombinant viruses containing swapped polymerase genes in mice.

<p>(A) Six-week-old female BALB/c mice (n = 15) were infected intranasally with 2×10³ TCID₅₀ of recombinant viruses. At the indicated time points, the infected mice (n = 5) were euthanized, and the viral titers in lungs were determined using MDCK cells (*, <i>p<0</i>.<i>01</i>). (B) Representative histopathological changes in H&E-stained lung tissues from mice infected with recombinant viruses at 5 dpi (20×).</p

Pathogenicity of wt HB04 and HN05 viruses.

<p>(A) Survival rate of mice after intranasal inoculation with10⁵ TCID₅₀ of the HB04 (n = 5) or HN05 (n = 5) virus. Mortality was monitored daily for 14 dpi. (B) Mean ± standard deviation (SD) of the percent body weight change of groups of mice (n = 5) after inoculation. (C) Mean viral titers ± SD in mouse-lung homogenates (n = 5) at 1, 3, and 5 dpi (*, <i>p<0</i>.<i>01</i>). (D) Representative histopathological changes in H&E-stained lung tissues from mice infected with HB04 or HN05 at 5 dpi (20×).</p

Pathogenicity of rescued reassortant viruses in mice.

<p>Colored bars indicate viral gene segments. Segments derived from HB04 and HN05 are shown in blue and red, respectively. The MLD₅₀ of the rescued viruses was determined as described in the Materials and Methods section. The mean maximum weight loss was determined from five mice (percentage weight loss relative to day 0 p.i.) after infection with 10⁵ TCID₅₀ of virus. The mean survival time (MST) of mice infected with 10⁵ TCID₅₀ was calculated using the Kaplan-Meier method.</p

Amino acid differences between the H5N1 influenza strains HB04 and HN05.

<p>Amino acid differences between the H5N1 influenza strains HB04 and HN05.</p

Evaluation of Preoperative Hematologic Markers as Prognostic Factors and Establishment of Novel Risk Stratification in Resected pN0 Non-Small-Cell Lung Cancer

<div><p>Background</p><p>The aims of this study were to investigate whether the preoperative hematologic markers, the neutrophil-lymphocyte ratio (NLR) or the platelet-lymphocyte ratio (PLR) were prognostic indicators and to develop a novel risk stratification model in pN0 non-small-cell lung cancer (NSCLC).</p><p>Methods</p><p>We performed a retrospective analysis of 400 consecutive pN0 NSCLC patients. Prognostic values were evaluated by Cox proportional hazard model analyses and patients were stratified according to relative risks for patients’ survival.</p><p>Results</p><p>During the follow-up, 117 patients had cancer recurrence, and 86 patients died. In univariate analysis, age, gender, smoke status and tumor size as well as WBC, NEU, LYM, PLR and NLR were significantly associated with patients’ prognosis. In multivariate analysis, age, tumor size and NLR were independent predictors for patients’ overall survival (P = 0.024, 0.001, and 0.002 respectively). PLR didn’t associated with patients’ survival in multivariate analysis. Patients were stratified into 3 risk groups and the differences among the groups were significant according to disease free survival and overall survival (P = 0.000 and 0.000 respectively).</p><p>Conclusions</p><p>We confirmed that NLR other than PLR was an independent prognostic factor. Combination of NLR, age and tumor size could stratify pN0 NSCLC patients into 3 risk groups and enabled us to develop a novel risk stratification model.</p></div

Distribution of clinical characteristics stratified by pretreatment NLR or PLR.

<p>Distribution of clinical characteristics stratified by pretreatment NLR or PLR.</p

Kaplan-Meier estimates according to low, intermediate and high risk groups on DFS (a) and OS (b).

<p>Kaplan-Meier estimates according to low, intermediate and high risk groups on DFS (a) and OS (b).</p

Clinical characteristic of all 400 lung cancer patients.

<p>Clinical characteristic of all 400 lung cancer patients.</p