Global map of survey responses showing presence reports of MNF from the survey and confirmed locations from the existing literature.

<p>Global map of survey responses showing presence reports of MNF from the survey and confirmed locations from the existing literature.</p

Presence observations of MNF by region and work sector of respondent.

<p>Presence observations of MNF by region and work sector of respondent.</p

Proportion of observations citing various drivers for people engaging and factors that may influence people not to MN fish.

<p>Proportion of observations citing various drivers for people engaging and factors that may influence people not to MN fish.</p

Cumulative first observations of MNF by region.

<p>Black line represents Global cumulative number of Long Lasting Insecticide-treated nets (LLINs) distributed since launch of Roll Back Malaria Programme, net data sourced from The Alliance for Malaria Prevention Net Mapping Project (2004-present) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191519#pone.0191519.ref021" target="_blank">21</a>].</p

Unraveling endothelin-1 induced hypercontractility of human pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension

<div><p>Contraction of human pulmonary artery smooth muscle cells (HPASMC) isolated from pulmonary arterial hypertensive (PAH) and normal (non-PAH) subject lungs was determined and measured with real-time electrical impedance. Treatment of HPASMC with vasoactive peptides, endothelin-1 (ET-1) and bradykinin (BK) but not angiotensin II, induced a temporal decrease in the electrical impedance profile mirroring constrictive morphological change of the cells which typically was more robust in PAH as opposed to non-PAH cells. Inhibition with LIMKi3 and a cofilin targeted motif mimicking cell permeable peptide (MMCPP) had no effect on ET-1 induced HPASMC contraction indicating a negligible role for these actin regulatory proteins. On the other hand, a MMCPP blocking the activity of caldesmon reduced ET-1 promoted contraction pointing to a regulatory role of this protein and its activation pathway in HPASMC contraction. Inhibition of this MEK/ERK/p90RSK pathway, which is an upstream regulator of caldesmon phosphorylation, reduced ET-1 induced cell contraction. While the regulation of ET-1 induced cell contraction was found to be similar in PAH and non-PAH cells, a key difference was the response to pharmacological inhibitors and to siRNA knockdown of Rho kinases (ROCK1/ROCK2). The PAH cells required much higher concentrations of inhibitors to abrogate ET-1 induced contractions and their contraction was not affected by siRNA against either ROCK1 or ROCK2. Lastly, blocking of L-type and T-type Ca²⁺ channels had no effect on ET-1 or BK induced contraction. However, inhibiting the activity of the sarcoplasmic reticulum Ca²⁺ ATPase blunted ET-1 and BK induced HPASMC contraction in both PAH and non-PAH derived HPASMC. In summary, our findings here together with previous communications illustrate similarities and differences in the regulation PAH and non-PAH smooth muscle cell contraction relating to calcium translocation, RhoA/ROCK signaling and the activity of caldesmon. These findings may provide useful tools in achieving the regulation of the vascular hypercontractility taking place in PAH.</p></div

Effect of vascular effectors, bradykinin and angiotensin, on HPASMC contraction.

<p>A representative HPASMC sample (Control-5) showing the average normalized cell index of triplicate wells treated without (CON) or with 100 nM bradykinin (BK) or 100 nM angiotensin II (AT) (A). Bar graph where bar heights represent average relative cell index at negative peak for n = 4 HPASMC strains. The error bars represent standard deviation. Relative cell index at negative peak was calculated by dividing the minimum cell index of the treatment group (AT, BK or ET-1) by the control group (CON) at that same time point. This was done for each HPASMC cell strain. Then, an average and standard deviation were calculated across n = 4 biological replicates. * p< 0.01 vs CON.</p

Detection of ET-1 induced contraction in HPASMC by electrical impedance.

<p>Electrical impedance was measured as described in the methods section. Triplicate wells were used with or without (CON) 1 nM, 10 nM or 100 nM ET-1 (A). The line graphs represent the average normalized cell index across triplicate wells and the error bars are the standard deviation at each time point. The bar graphs represent the average negative peak for each triplicate and the error bars are the standard deviation at that time point. Triplicate wells treated with vehicle (CON), 1 uM BQ123 plus 10 nM ET-1, 10 nM ET-1 or 10 nM of sarafotoxin (SF6C) were measured as well (B). This figure illustrates a non-PAH HPASMC (Control-1) strain that is representative of results obtained from various HPASMC strains. * p< 0.01 vs CON; ** p< 0.001 vs CON.</p

Dot plot comparing ET-1 induced contraction magnitudes in non-PAH and PAH HPASMC.

<p>Contraction magnitudes were calculated for non-PAH (n = 5) and PAH (n = 5) HPASMC by taking the maximum negative peak value of each sample and dividing by one of the non-PAH samples giving a relative cell index value. Vertical dots indicate relative cell index for each non-PAH (triangle) and PAH (circle) HPASMC sample. The horizontal line represents the median of the group. A Welch’s two sample t-test assuming unequal variance was performed between the non-PAH and PAH groups; p-value = 0.055.</p

Effect of ROCK inhibitor on the ET-1 induced contraction in non-PAH and PAH HPASMC.

<p>Representative experiments showing triplicate wells which were pretreated without or with 1, 10 uM or 25 uM Y27632 (ROCKin) for 1 h and then with or without ET-1 in non-PAH (A) and PAH (B) HPASMC. These values at the negative peak of the cell index are illustrated with bar graphs with error bars representing standard deviations. #p< 0.05 vs CON.</p

The effect of calcium channel blockers on ET-1 and bradykinin induced cell contraction.

<p>Representative experiments showing bar graphs with standard deviation error bars for the effects of 10 uM nifedipine (Nif), 10 uM thapsigargin (thaps) (A, C), or mibefradil (Mib) (B, D) on ET-1 (A, B) or bradykinin (BK) (C, D) induced cell contraction. All inhibitors were pre-incubated with the cells for 1 h before addition of ET-1 or BK. #p< 0.05 vs CON.</p