Genome Wide Expression Analysis Suggests Perturbation of Vascular Homeostasis during High Altitude Pulmonary Edema

<div><p>Background</p><p>High altitude pulmonary edema (HAPE) is a life-threatening form of non-cardiogenic edema which occurs in unacclimatized but otherwise normal individuals within two to four days after rapid ascent to altitude beyond 3000 m. The precise pathoetiology and inciting mechanisms regulating HAPE remain unclear.</p><p>Methodology/Principle findings</p><p>We performed global gene expression profiling in individuals with established HAPE compared to acclimatized individuals. Our data suggests concurrent modulation of multiple pathways which regulate vascular homeostasis and consequently lung fluid dynamics. These pathways included those which regulate vasoconstriction through smooth muscle contraction, cellular actin cytoskeleton rearrangements and endothelial permeability/dysfunction. Some notable genes within these pathways included <i>MYLK</i>; rho family members <i>ARGEF11</i>, <i>ARHGAP24</i>; cell adhesion molecules such as <i>CLDN6</i>, <i>CLDN23</i>, <i>PXN</i> and <i>VCAM1</i> besides other signaling intermediates. Further, several important regulators of systemic/pulmonary hypertension including <i>ADRA1D, ECE1,</i> and <i>EDNRA</i> were upregulated in HAPE. We also observed significant upregulation of genes involved in paracrine signaling through chemokines and lymphocyte activation pathways during HAPE represented by transcripts of <i>TNF</i>, <i>JAK2, MAP2K2, MAP2K7, MAPK10</i>, <i>PLCB1</i>, <i>ARAF</i>, <i>SOS1</i>, <i>PAK3</i> and <i>RELA</i> amongst others. Perturbation of such pathways can potentially skew vascular homeostatic equilibrium towards altered vascular permeability. Additionally, differential regulation of hypoxia-sensing, hypoxia-response and OXPHOS pathway genes in individuals with HAPE were also observed.</p><p>Conclusions/Significance</p><p>Our data reveals specific components of the complex molecular circuitry underlying HAPE. We show concurrent perturbation of multiple pathways regulating vascular homeostasis and suggest multi-genic nature of regulation of HAPE.</p></div

Additional file 1 of Investigating the plant growth promoting and biocontrol potentiality of endophytic Streptomyces SP. SP5 against early blight in Solanum lycopersicum seedlings

Additional file 1

Schematic representation of possible events occurring during HAPE.

<p>The pathways suggested by the current data set have been integrated with established phenomenon such as pulmonary vasoconstriction, elevated pulmonary artery pressure which are known to precede HAPE. Perturbation of pathways such as those regulating vasoconstriction, inflammation, gap junctions and adhesion molecules can dysregulate vascular homeostasis leading to fluid leak and edema formation.</p

Comparison of relative expression (log2 values) of selected genes obtained by microarray and real time PCR experiments (TLDA).

<p>17 out of 20 genes show similarity of trend on both the platforms.</p

Hierarchical clustering of gene expression data obtained from acclimatized controls (CN) and HAPE (PE) individuals.

<p>Hierarchical clustering distinctly separated the two groups of individuals (CN and PE) indicating unique gene expression signatures. Expression values of specific genes are represented by color intensities shown in the reference color key.</p

Work flow of gene expression analysis.

<p>The figure represents scheme of extraction of differentially expressed genes (up regulated: 420, down regulated: 308) from the raw microarray data.</p

KEGG Pathway-specific gene transcripts in HAPE data set (Extracted from DAVID Bioinformatics Resource).

<p>List of key KEGG Pathways extracted from differentially expressed genes utilizing DAVID Bioinformatic Resource. Specific pathways are arranged in order of their p-value. Gene names and Fold changes for individual genes were added from the original data set.</p

Clustering of GO terms related to biological processes.

<p>GO terms were clustered utilizing software BINGO and individual clusters indicated as shown in the figure.</p

<i>N</i>-(4-Hydroxyphenyl)-3,4,5-trimethoxybenzamide derivatives as potential memory enhancers: synthesis, biological evaluation and molecular simulation studies

<p>The present paper describes the synthesis, biological evaluation and molecular simulation studies of a series of <i>N</i>-(4-hydroxyphenyl)-3,4,5-trimethoxybenzamide derivatives with <i>N,N</i>-dialkylaminoethoxy/propoxy moiety as potential memory enhancers with acetylcholinesterase-inhibiting activity having IC₅₀ in low micromolar range (4.0–16.5 μM). All the compounds showed a good degree of agreement between <i>in vivo</i> and <i>in vitro</i> results as most of these derivatives showed dose-dependent increase in percent retention. Compound <b>10a</b> showed significant % retention of 84.73 ± 4.51 as compared to piracetam (46.88 ± 5.42) at 3 mg kg⁻¹ and also exhibited a maximal percent inhibition of 97% at 50 μM. Molecular docking, MM-GBSA and molecular simulation studies were performed establishing a correlation between the experimental biology and <i>in silico</i> results. <i>In silico</i> results indicate that all the compounds have better docking scores and predicted binding free energies as compared to cocrystallized ligand with the best potent ligand retaining conserved hydrophobic interactions with residues of catalytic triad (HIS447), catalytic anionic site (CAS) (TRP86, TYR337, PHE338) and peripheral anionic site (PAS) (TYR72, TYR124, TRP286 and TYR341). Root mean square deviation (RMSD = 2.4 Å) and root mean square fluctuations of <b>10a</b>–AChE complex during simulation proved its stable nature in binding toward acetylcholinesterase. The docked conformation of <b>10a</b> and other analogs at the binding site have also been simulated with polar and nonpolar interactions interlining the gorge residues from PAS to catalytic triad.</p

Human Immunodeficiency Virus 1 Preferentially Fuses with pH-Neutral Endocytic Vesicles in Cell Lines and Human Primary CD4+ T‑Cells

Despite extensive efforts, the principal sites of productive HIV-1 entry in different target cellsplasma membrane (PM) vs endosomesremain controversial. To delineate the site(s) of HIV-1 fusion, we implemented a triple labeling approach that involves tagging pseudoviruses with the fluid-phase viral content marker, iCherry, the viral membrane marker, DiD, and the extraviral pH sensor, ecliptic pHluorin. The viral content marker iCherry is released into the cytoplasm upon virus–cell fusion irrespective of the sites of fusion. In contrast, the extent of dilution of the membrane marker upon fusion with the PM (loss of signal) vs the endosomal membrane (no change in punctate DiD appearance) discriminates between the principal sites of viral fusion. Additionally, ecliptic pHluorin incorporated into the viral membrane reports whether virus fusion occurs in acidic endosomes. Real-time single virus imaging in living HeLa-derived cells, a CD4+ T-cell line, and activated primary human CD4+ T-cells revealed a strong (80–90%) HIV-1 preference for fusion with endosomes. Intriguingly, we observed HIV-1 fusion only with pH-neutral intracellular vesicles and never with acidified endosomes. These endocytic fusion events are likely culminating in productive infection since endocytic inhibitors, such as EIPA, Pitstop2, and Dynasore, as well as a dominant-negative dynamin-2 mutant, inhibited HIV-1 infection in HeLa-derived and primary CD4+ T-cells. Furthermore, the inhibition of endocytosis in HeLa-derived cells promoted hemifusion at the PM but abrogated complete fusion. Collectively, these data reveal that the primary HIV-1 entry pathway in diverse cell types is through fusion with pH-neutral intracellular vesicles