Pumping Small Molecules Selectively through an Energy-Assisted Assembling Process at Nonequilibrium States

In living organisms, precise control over the spatial and temporal distribution of molecules, including pheromones, is crucial. This level of control is equally important for the development of artificial active materials. In this study, we successfully controlled the distribution of small molecules in the system at nonequilibrium states by actively transporting them, even against the apparent concentration gradient, with high selectivity. As a demonstration, in the aqueous solution of acid orange (AO7) and TMC10COOH, we found that AO7 molecules can coassemble with transient anhydride (TMC10CO)2O to form larger assemblies in the presence of chemical fuel 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC). This led to a decrease in local free AO7 concentration and caused AO7 molecules from other locations in the solution to move toward the assemblies. Consequently, AO7 accumulates at the location where EDC was injected. By continuously injecting EDC, we could maintain a stable high value of the apparent AO7 concentration at the injection point. We also observed that this process which operated at nonequilibrium states exhibited high selectivity

The effect of neonatal IA treatment on psychological behavior of adult rats.

<p><b>A... </b><b><i>Sucrose consumption test</i></b>. The percentage of animals with sucrose consumption ≥75% was significantly lower in IA-treated rats as compared with control rats (*P<0.05) (see text for details). <b>B... </b><b><i>Forced swimming test</i></b>. Immobility was significantly increased (P<0.001) and the climbing and swimming were significantly reduced (P<0.001 and P<0.05) in IA-treated rats compared with control rats (see text for details). Data represent the mean ± SEM of 20 rats per group. <b>C... </b><b><i>Elevated plus maze test</i></b>. <b>i.</b> The total distance traveled (cm) in the 5-minute test time was not different between the IA-treated and control groups. <b>ii.</b> The percentage distance traveled in open arm was significantly reduced in IA-treated rats.</p

The effect of neonatal IA treatment on psychological behavior of adult rats (continued).

<p><b>A.. </b><b><i>Open field test</i></b>. <b>i.</b> The total distance traveled (cm) in the 30-minute test time. <b>ii.</b> The percentage time spent in center. There was a significant decrease in the time that IA-treated rats spent in the center as well as the total distance traveled. Data represent the mean ± SEM of 20 rats per group. <b>B.. </b><b><i>Light dark box test</i></b>. <b>i.</b> The percent of time spent in the light box was significantly shorter in the IA-treated rats as compared with control rats. <b>ii.</b> The number of light-dark box transitions was not significantly different between the two groups (n = 10 per group). (* =  Significantly different from control group).</p

ACTH and corticosterone responses to minor stress (saline injection).

<p><b>A.</b> Saline injection significantly increased ACTH secretion in IA-treated rats (* P<0.05 compared with stress control rats; <b>&</b> P<0.05 compared with non-stress IA-treated rats). <b>B.</b> The basal level of corticosterone was increased in IA treated rats than control rats (<b>&</b> P<0.05 compared with non-stress control rats; * P<0.05 stress rats compared with non-stress rats) (see text for details). Data represent the mean ± SEM of 8 rats per group. (* =  Significantly different from control group) <b>C.</b> Representative Photographs (Magnification: 10x) of CRF change in paraventricular nucleus (PVN) and central nucleus of the amgdyla (CeA). <b>D.</b> Change of CRF staining Density in PVN and CeA area. Data represent the mean ± SEM of 3 sections per rat, 4 rats per group. (* =  Significantly different from control group).</p

Gene silencing and mortality rate of <i>H. longicornis</i> 96 hours after a single dsRNA injection.

<p>dsRNA complementary to <i>HlSRB</i>, <i>HlVgR</i>, and <i>HlVg-1</i> was injected into <i>H. longicornis</i> adult females. The injected ticks were allowed to rest at 25°C in an incubator for four days to check mortality rates and gene silencing. RT-PCR analysis (A). PCR was performed using cDNA synthesized from three ticks injected with <i>HlSRB</i>, <i>HlVgR</i>, <i>HlVg-1</i>, or <i>luc</i> dsRNA with primer sets specific to <i>HlSRB</i>, <i>HlVgR</i>, <i>HlVg-1</i>, and the <i>β-actin</i> gene. Lane 1, <i>HlSRB</i> dsRNA-injected ticks; lanes 2, 4, and 6, <i>luc</i> dsRNA-injected ticks; lane 3, <i>HlVgR</i> dsRNA-injected ticks; lane 5, <i>HlVg-1</i> dsRNA-injected ticks. Mortality rates (B). Each panel represents treatment with one gene-specific dsRNA. Mortality rates were calculated by the percentage of number of dead ticks to the number of ticks used at the beginning of experiment in a different time course. <i>HlSRB</i>, <i>HlSRB</i> dsRNA-injected ticks; <i>HlVgR</i>, <i>HlVgR</i> dsRNA-injected ticks; <i>HlVg-1</i>, <i>HlVg-1</i> dsRNA-injected ticks; <i>luciferase</i>, <i>luciferase</i> dsRNA-injected ticks.</p

Effects of intragastric RTX and antalarmin.

<p><b>A: Gastric afferent activity after intragastric RTX as compared to vehicle treatement. </b><b>A.i:</b> There is a significant (p<0.05 using ANOVA) decline in basal splanchnic nerve activity after RTX but not in vehicle treated group. <b>A.ii:</b> There is a significant (p<0.05 using ANOVA) decline in the evoked response to gastric distention, GD, at 80 mmHg of pressure) 30 minutes after RTX but no change in the vehicle treated group (n =  4 each). (*  =  Significantly different than Pre-treatment baseline using Bunnet multiple comparison test). <b>B: Effects of gastric sensory ablation on depression-like behavior.</b> No differences were seen in the results of the forced swimming test in IA-treated animals that underwent intragastric RTX treatment as compared with animals that received vehicle only. <b>C. Effects of the CRF1 antagonist antalarmin on depression-like behavior.</b> Antalarmin resulted in a significant reduced in immobility and an increase in the climbing and swimming in IA-treated rats compared with vehicle treated IA-treated rats.</p

Expression profiles of HlSRB, HlVg-1, and HlVgR genes and proteins in different tissues of <i>H. longicornis</i>.

<p>Individually or in combination of <i>HlSRB</i>, <i>HlVg-1</i>, <i>HlVgR</i>, and <i>luc</i> dsRNA(s) were injected into <i>H. longicornis</i> adult ticks. The midguts and ovaries of dsRNA-injected ticks at 4 days of feeding were dissected out in 0.1% diethylpyrocarbonate-treated 1 × PBS (-) under a microscope. The name of each dsRNA group is indicated above. RT-PCR analysis and Western blot analysis were conducted using the midguts (A and B) and ovaries (C and D).</p

Silencing of HlSRB, HlVgR, and HlVg-1 genes and proteins in the whole body of <i>H. longicornis.</i>

<p>Individually or in combination of <i>HlSRB</i>, <i>HlVgR</i>, <i>HlVg-1</i>, and <i>luc</i> dsRNA(s) were injected into <i>H. longicornis</i> adult ticks. The injected ticks were left for 12 hours at 25°C and infested on the rabbits for four days and then ticks samples were collected for RNA extraction and the preparation of ticks protein lysates in each group. The name of each dsRNA group is indicated above. RT-PCR analysis (A). RT-PCR analysis was performed as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028407#pone-0028407-g001" target="_blank">Fig. 1</a>. (A). Western blot analysis (B). Tick lysates were subjected to SDS-PAGE under reducing conditions and transferred to a PVDF membrane. The membrane was probed with mouse anti-rHlSRB, anti-rHlVgR, or anti-rHlVg-1 sera; mouse anti-actin serum was used as a control. The name of each dsRNA group is the same as that used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028407#pone-0028407-g001" target="_blank">Fig. 1</a>.</p

Confirmation of RNAi on the endogenous HlSRB, HlVg-1, and HlVgR in the different tissues of <i>H. longicornis</i>.

<p>The dissected tissues from the dsRNA-injected 4-days-feeding ticks were observed under fluorescence microscopy. The name of each dsRNA group is indicated above. The midguts were stained with anti-rHlSRB and anti-rHlVg-1 antibodies followed by Alexa 488-conjugated mouse anti-IgG with DAPI (A). Arrowheads indicate the native HlSRB and HlVg-1 expressed in the midguts. ML, midgut lumen; MC, midgut cells. The ovaries, staining pattern of anti-rHlSRB and anti-rHlVgR serum were used as first antibodies (B). The mouse anti-IgG conjugated with Alexa 488 was used as a second antibody with DAPI for the upper panels and Alexa 594 with Propidium Iodide for the lower panels. Arrowheads indicate the native HlSRB and HlVgR expressed in the ovaries. OO, oocyte; OD, oviduct. The <i>scale bar</i> represents 20 µm.</p

<b>Table 1.</b> Female tick groups injected with a single and a combination of dsRNA(s).

<p>The first and second dsRNA injections were carried out at 96-hours interval.</p