Horticultural markets promote alien species invasions : an Estonian case study of herbaceous perennials

Gardening is a popular pastime, but commercial horticulture is responsible for the introduction of alien species and contributes to invasions in a variety of ways. Although an extensive international literature is available on plant invasions, it is still important at the national level to examine the influence of local factors. Accordingly, 17 nurseries in Estonia that cultivated and sold perennial alien species were selected, and a list of species and prices was compiled. The relationships between species status, and factors such as their abundance in the wild were examined statistically. A qualitative list of the nationally problematic species among herbaceous perennials was also completed. A total of 880 taxa were recorded, of which 10.3% were native and 89.7% alien. In all, 87.3% of the alien species were still confined to cultivated areas. The ecological and socio-economic characteristics of the taxa were described, and lists of the families of casual, naturalised and invasive aliens were provided. Both native and increasing wild alien species have a very similar profile on the market. Alien species that are less expensive, widely available and have more cultivars per species on the market are also more likely to escape. The invasive status and abundance of escaped aliens in an area increases with residence time. In general, socio-economic factors create new and reflect previous propagule pressures from commercial horticulture, which continuously increase the likelihood of alien species surviving and invading new areas. Our findings suggest that these national socioeconomic market-related factors explain much of the invasiveness of various perennial ornamental species, and therefore regional and national authorities urgently need to regulate and control the ornamental plant trade to diminish the risk of new invasions

Hantavirus-infection Confers Resistance to Cytotoxic Lymphocyte-Mediated Apoptosis

<div><p>Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardio-pulmonary syndrome (HCPS; also called hantavirus pulmonary syndrome (HPS)), both human diseases with high case-fatality rates. Endothelial cells are the main targets for hantaviruses. An intriguing observation in patients with HFRS and HCPS is that on one hand the virus infection leads to strong activation of CD8 T cells and NK cells, on the other hand no obvious destruction of infected endothelial cells is observed. Here, we provide an explanation for this dichotomy by showing that hantavirus-infected endothelial cells are protected from cytotoxic lymphocyte-mediated induction of apoptosis. When dissecting potential mechanisms behind this phenomenon, we discovered that the hantavirus nucleocapsid protein inhibits the enzymatic activity of both granzyme B and caspase 3. This provides a tentative explanation for the hantavirus-mediated block of cytotoxic granule-mediated apoptosis-induction, and hence the protection of infected cells from cytotoxic lymphocytes. These findings may explain why infected endothelial cells in hantavirus-infected patients are not destroyed by the strong cytotoxic lymphocyte response.</p> </div

Hantaviruses inhibit staurosporine-induced apoptosis.

<p>Endothelial and epithelial cells were infected with hantavirus for three days or left uninfected (control). Cells were then exposed to the apoptosis-inducing drug staurosporine, and subsequently analyzed for induction of apoptosis. (<b>A and B</b>) Percentage of TUNEL-positive endothelial cells (A) and A549 cells (B) infected with ANDV or HTNV for three days and then treated with staurosporine for four hours. A low level of ANDV or HTNV (MOI 0.01 for both) was used for initial infection to allow for a level of approximately 20% infected cells at three days pi, the time at which cells were exposed to staurosporine. Amounts of TUNEL-positive infected and uninfected cells on the same slides were then determined using fluorescence microscopy. Staurosporine-treated uninfected cells represent maximal level of TUNEL-positive cells. Data shown represent the mean ± SEM of three independent experiments carried out in duplicate. One-way ANOVA was used for statistical evaluation; ** p<0.01; *** p<0.001. (<b>C</b>) Western blot analyses of pro-caspase 3 and caspase 3 from lysate of staurosporine-treated ANDV-infected and uninfected A549 cells. ANDV nucleocapsid protein (ANDV-N) was visualized with the mAb 7B3/F7. Calnexin was used as a control to show that similar amounts of protein were loaded in all wells. One representative experiment out of three is shown. Band intensity was analyzed by densitometry of cleaved, active, caspase 3 and full-length, inactive, pro-caspase 3. The caspase 3/pro-caspase 3-ratio was then calculated and compared between infected and uninfected cells. Staurosporine-treated uninfected cells represent the maximal caspase 3/pro-caspase 3 ratio at the indicated time-points after start of staurosporine-treatment. Data shown represent the mean ± SEM of three independent experiments. Two-way ANOVA was used for statistical evaluation; *** p<0.001. STS; staurosporine. (<b>D</b>) Caspase 3-activity after staurosporine-treatment of ANDV or HTNV infected A549 cells compared to that detected in uninfected A549 cells at the same time points. Levels of total protein in the samples were measured using a Bradford assay. Staurosporine-treated uninfected cells represent maximal level of caspase 3-activity/mg of total cellular protein. Data shown represent the mean ± SEM of three independent experiments carried out in duplicate. Two-way ANOVA was used for statistical evaluation; * p<0.05; ** p<0.01; *** p<0.001. STS; staurosporine. (<b>E</b>) Western blot analyses of full-length PARP and of caspase 3-cleaved PARP in staurosporine-treated uninfected and ANDV-infected A549 cells. ANDV-N was visualized with the mAb 7B3/F7. Calnexin was used as a control to show that similar amounts of protein were loaded in all wells. One representative experiment out of three is shown. Band intensity was analyzed by densitometry of caspase 3-cleaved PARP and full-length PARP. The caspase 3-cleaved PARP/PARP-ratio was then calculated and compared between infected and uninfected cells. Staurosporine-treated uninfected cells represent maximal caspase 3-cleaved PARP/full-length PARP-ratio at the indicated time-points after start of staurosporine-treatment. Data shown represent the mean ± SEM of three independent experiments. Two-way ANOVA was used for statistical evaluation; * p<0.05. STS; staurosporine.</p

ANDV nucleocapsid protein inhibits caspase 3-activity.

<p>Analysis of possible interactions between ANDV nucleocapsid protein and caspase 3. (<b>A</b>) Western blot analyses showing a cleaved fragment of the ANDV nucleocapsid protein (ANDV-N) after incubation with recombinant caspase 3 in the presence or absence of the caspase 3-inhibitor DEVD-CHO. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. (<b>B</b>) ANDV-infected A549 cells were left untreated or treated with staurosporine, in the presence or absence of the caspase-inhibitor Z-VAD-fmk. Lysed cells were then subjected to Western blot analyses to visualize cleavage of the viral ANDV nucleocapsid protein (ANDV-N) after staurosporine-treatment. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. (<b>C</b>) Caspase 3-activity was measured after pre-incubation of recombinant caspase 3 with recombinant ANDV nucleocapsid protein (rANDV-N) or with control protein (rDHFR) for 30 minutes. Level of caspase 3-activity after co-incubation with rDHFRS represent maximal caspase 3 activity. Level of caspase 3-activity after co-incubation of caspase 3 with rANDV-N was compared with caspase 3-activity after co-incubation with rDHFRS. Data shown are mean ± SEM of three independent experiments carried out in duplicate. Two-tailed Student's t test was used for statistical evaluation; * p<0.05. (<b>D</b>) Caspase 3-activity after pre-incubation for 30 minutes with increasing amount of recombinant ANDV nucleocapsid protein (rANDV-N). Data shown are mean ± SEM of three independent experiments carried out in duplicate. One-way ANOVA was used for statistical evaluation; p<0.05 for 1 µg versus 0.5 µg and for 1 µg versus 0.25 µg. (<b>E</b>) Percentage of TUNEL-positive A549 cells transfected with PCMV-bios (control), PCMV-bios-ANDV-N-wt (ANDV-N-wt), or PCMV-bios-ANDV-N Asp285 to Ala285 mutant (ANDV-N-mut) for 20 hours, and then treated with staurosporine for four hours. TUNEL-positive cells were identified using fluorescence microscopy. Staurosporine-treated cells transfected with empty plasmid represent maximal level of TUNEL-positive cells. Data shown are mean ± SEM of three independent experiments carried out in duplicate. One-way ANOVA was used for statistical evaluation; * p<0.05; ** p<0.01. N, nucleocapsid protein; wt, wild-type; mut, mutated. (<b>F</b>) Percentage of caspase 3-positive A549 cells transfected with PCMV-bios (control), PCMV-bios-ANDV-N-wt (ANDV-N-wt), or PCMV-bios-ANDV-N Asp285 to Ala285 mutant (ANDV-N-mut) for 20 hours, and then treated with staurosporine for four hours. Caspase 3-positive cells, positive for FLICA-staining, were identified using fluorescence microscopy. Staurosporine-treated cells transfected with empty plasmid represent maximal level of caspase 3-positive cells. Data shown are mean ± SEM of three independent experiments carried out in duplicate. One-way ANOVA was used for statistical evaluation; *** p<0.001. N, nucleocapsid protein; wt, wild-type; mut, mutated.</p

Hantavirus-infection inhibits NK cell-mediated activation of caspase 3 in endothelial cells.

<p>Prevention of caspase 3 activation in infected cells exposed to IL-2-activated NK cells. (<b>A</b>) Representative flow cytometry histogram showing cellular caspase 3-activity in uninfected and HTNV-infected HLA class I blocked endothelial cells with and without co-incubation with IL-2-activated NK cells. Data shown is one representative donor out of six. (<b>B</b>) Percentage of caspase 3-positive uninfected and HTNV-infected endothelial cells after co-incubation with IL-2-activated NK cells analyzed by flow cytometry. Data shown represent two independent experiments from six donors. Two-tailed Student's <i>t</i> test was used for statistical evaluation; ** p<0.01.</p

ANDV nucleocapsid protein inhibits granzyme B-activity.

<p>Analysis of possible interactions between ANDV nucleocapsid protein and granzyme B. (<b>A</b>) Western blot analyses showing cleaved fragments of the ANDV nucleocapsid protein (ANDV-N) after incubation with recombinant granzyme B. Full-length and cleaved ANDV-N was visualized with the mAb 7B3/F7. One representative experiment out of three is shown. (<b>B</b>) Granzyme B-activity was measured after pre-incubation of recombinant granzyme B with recombinant ANDV nucleocapsid protein (rANDV-N) for 30 minutes. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003272#s2" target="_blank">Results</a> were compared to granzyme B-activity for granzyme B pre-incubated with control protein (rDHFR). Activity after co-incubation with rDHFRS represents maximal granzyme B-activity. Data shown are mean ± SEM of three independent experiments carried out in duplicate. Two-tailed Student's <i>t</i> test was used for statistical evaluation; *** p<0.001. (<b>C</b>) Granzyme B-activity after pre-incubation for 30 minutes with increasing amount of recombinant ANDV nucleocapsid protein (rANDV-N). Data shown are mean ± SEM of three independent experiments carried out in duplicate. One-way ANOVA was used for statistical evaluation: except for 1 µg versus 0.75 µg (non significant), p<0.001 for all other comparisons. (<b>D</b>) Granzyme B-activity after pre-incubation for 0.5–27 hours of recombinant granzyme B with recombinant ANDV nucleocapsid protein (rANDV-N) compared to control protein (rDHFR). Data shown are from one experiment carried out in duplicate.</p

Multicenter Evaluation of the Idylla GeneFusion in Non-Small-Cell Lung Cancer.

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods

Multicenter Evaluation of the Idylla GeneFusion in Non-Small-Cell Lung Cancer

Targeted therapy in lung cancer requires the assessment of multiple oncogenic driver alterations, including fusion genes. This retrospective study evaluated the Idylla GeneFusion prototype, an automated and ease-of-use (<2 minutes) test, with a short turnaround time (3 hours) to detect fusions involving ALK, ROS1, RET, and NTRK1/2/3 genes and MET exon 14 skipping. This multicenter study (18 centers) included 313 tissue samples from lung cancer patients with 97 ALK, 44 ROS1, 20 RET, and 5 NTRKs fusions, 32 MET exon 14 skipping, and 115 wild-type samples, previously identified with reference methods (RNA-based next generation sequencing/fluorescence in situ hybridization/quantitative PCR). Valid results were obtained for 306 cases (98%), overall concordance between Idylla and the reference methods was 89% (273/306); overall sensitivity and specificity were 85% (165/193) and 96% (108/113), respectively. Discordances were observed in 28 samples, where Idylla did not detect the alteration identified by the reference methods; and 5 samples where Idylla identified an alteration not detected by the reference methods. All of the ALK-, ROS1-, and RET-specific fusions and MET exon 14 skipping identified by Idylla GeneFusion were confirmed by reference method. To conclude, Idylla GeneFusion is a clinically valuable test that does not require a specific infrastructure, allowing a rapid result. The absence of alteration or the detection of expression imbalance only requires additional testing by orthogonal methods