Screening of Random Peptide Library of Hemagglutinin from Pandemic 2009 A(H1N1) Influenza Virus Reveals Unexpected Antigenically Important Regions

The antigenic structure of the membrane protein hemagglutinin (HA) from the 2009 A(H1N1) influenza virus was dissected with a high-throughput screening method using complex antisera. The approach involves generating yeast cell libraries displaying a pool of random peptides of controllable lengths on the cell surface, followed by one round of fluorescence-activated cell sorting (FACS) against antisera from mouse, goat and human, respectively. The amino acid residue frequency appearing in the antigenic peptides at both the primary sequence and structural level was determined and used to identify “hot spots” or antigenically important regions. Unexpectedly, different antigenic structures were seen for different antisera. Moreover, five antigenic regions were identified, of which all but one are located in the conserved HA stem region that is responsible for membrane fusion. Our findings are corroborated by several recent studies on cross-neutralizing H1 subtype antibodies that recognize the HA stem region. The antigenic peptides identified may provide clues for creating peptide vaccines with better accessibility to memory B cells and better induction of cross-neutralizing antibodies than the whole HA protein. The scheme used in this study enables a direct mapping of the antigenic regions of viral proteins recognized by antisera, and may be useful for dissecting the antigenic structures of other viral proteins

Effect of carbon monoxide-releasing molecules II-liberated CO on suppressing inflammatory response in sepsis by interfering with nuclear factor kappa B activation.

Sepsis continues to be a challenge in clinic. The rates of mortality in sepsis patients remain high. The present study aimed to investigate the effects and the underlying mechanisms of carbon monoxide-releasing molecules II (CORM-2)-liberated CO on suppressing inflammatory response in sepsis. It was shown that treatment of septic mice with CORM-2 attenuated PMN accumulation, downregulated cytokines production, inhibited expressions of iNOS and NF-κB activity in the lung and liver. In parallel, CORM-2 prevented activation of NF-κB in LPS-stimulated HUVEC. This was accompanied by a decrease in ROS and NO production, expression of ICAM-1 and subsequent PMN adhesion to HUVEC. These findings demonstrated that CORM-released CO attenuates inflammatory responses by interfering with NF-κB activation and therefore decreasing the expression of ICAM-1 and NO production, attenuating the oxidative stress and inflammation in sepsis

MOESM2 of Recombinant production of medium- to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag

Additional file 2. MALDI-TOF MS analyses of target peptides

MOESM1 of Recombinant production of medium- to large-sized peptides in Escherichia coli using a cleavable self-aggregating tag

Additional file 1. Peptide sequences

Effects of CORM-2 on protein expression of iNOS in the liver and lung of septic mice.

<p>Mice were challenged with CLP and treated with CORM-2 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075840#pone-0075840-g002" target="_blank">Figure 2</a>. Protein expression of iNOS in lung and liver was performed by western blotting at 24 h after CLP. A representative image is shown in A, the average optical density in three experiments are shown in B (lung) and C (liver), respectively. Results are mean ± SE. *<i>P</i><0.01 compared to sham mice. #<i>P</i><0.05 compared to CLP mice.</p

Effects of CORM-2 on NF-κB activation in LPS stimulated-HUVECs.

<p>HUVECs were grown to confluence in 48-well cell culture plates and were stimulated with LPS (10 µg/ml) for 4 h in the presence or absence of CORM-2 (10, 50, 100 µM). The NF-κB activation in HUVECs was measured by EMSA. The NF-κB activation was markedly increased in LPS-stimulated HUVECs and this increase was inhibited by administration of CORM-2 in a concentration dependent manner. A representative image is shown in A, the average optical density in three experiments are shown in B. All values are expressed as mean ± SE (<i>n</i> = 4). *<i>p</i><0.05 compared to control, #<i>p</i><0.05 compared to LPS.</p

Effects of CORM-2 on NF-κB activation in the lung and liver of septic mice.

<p>Mice were challenged with CLP and treated with CORM-2 as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075840#pone-0075840-g002" target="_blank">Figure 2</a>. Measurement of NF-κB activities were performed by mobility shift assay (EMSA) with ³² P-labeled NF-κB probe. The representative images are shown in A (lung) and C (liver), the average optical density in three experiments are shown in B (lung) and D (liver), respectively. All values are expressed as mean ± SE (<i>n</i> = 4). *<i>p</i><0.05 compared to sham, #<i>p</i><0.05 compared to CLP. Lane 1, sham; lane 2, CLP; Lane 3, CLP+iCORM; Lane 4, CLP+CORM.</p

Effect of CORM-2 on TNF-α and IL-6 levels in serum of septic mice.

<p>Mice were challenged with CLP and treated with CORM-2 or iCORM-2. Blood samples were obtained by cardiac puncture of the left ventricle. Concentrations of TNF-α and IL-6 levels were assayed by enzyme-linked immunosorbent assay kits. At 6 after CLP, TNF-α and IL-6 levels in serum of the CLP-challenged mice were markedly increased compared to the sham mice. This increase wasn’t abolished by administration of CORM-2 (A, B). However, the elevation in serum levels of TNF-α and IL-6 was significantly abolished by CORM-2 treatment at 12 and 24 h (C, D, E and F). Data are expressed as mean ± SE of three experiments. *<i>P</i><0.05 compared to sham group; #<i>P</i><0.05 compared to CLP group.</p

Effects of CORM-2 on intracellular production of ROS and NO in LPS-stimulated HUVECs.

<p>HUVECs were grown to confluence in 48-well cell culture plates and loaded with dihydrorhodamine 123 (DHR 123) or diaminofluorescein-FM (DAF-FM) for 1 h. Subsequently, HUVECs were stimulated with LPS (10 µg/ml) for 4 h in the presence or absence of CORM-2 (10, 50, 100 µM). Oxidative stress (DHR123 oxidation) (A) and NO production (DAF-FM nitration) (B) were assessed. All values are expressed as mean ± SE (<i>n</i> = 4). *<i>p</i><0.05 compared to control, #<i>p</i><0.05 compared to LPS. Note that LPS-induced oxidative stress and NO production were inhibited by CORM-2 in a dose-dependent manner.</p