Multiple alignment of the deduced amino acid sequences of E2F-2 from <i>P</i>. <i>monodon</i> and other species.

<p>Sequence logo representing the similarity is shown at the top of alignments and numbers of amino acid are listed on the right side of alignments. The GenBank numbers of E2F-2 are listed as follows: <i>P</i>. <i>monodon</i>: KY628943; <i>Apis mellifera</i>: XP_006561713.1; <i>Eufriesea mexicana</i>: OAD58031.1; <i>Linepithema humile</i>: XP_012219394.1; <i>Lasius niger</i>: KMQ93879.1; <i>Polistes canadensis</i> XP_014607688.1. E2F-TDP domain and coiled coil domain are indicated with black and green lines, respectively.</p

The gonadosomatic index (GSI, ovarian weight/body weight × 100) of shrimp after dsRNA-GFP-, dsRNA-E2F-2- and PBS injection.

<p>The gonadosomatic index (GSI, ovarian weight/body weight × 100) of shrimp after dsRNA-GFP-, dsRNA-E2F-2- and PBS injection.</p

Comparison of the genomic DNA sequence encoding E2F-2 in <i>P</i>. <i>monodon</i>.

<p>Green-shaded rectangles indicate exons, gray horizontal lines represent introns, and the numbers indicate exon and intron length (in bp).</p

Relative expression levels of <i>PmE2F-2</i> in various tissues detected by quantitative real-time PCR analysis using <i>EF-1α</i> as an internal reference.

<p>Vertical bars represented mean ±SD (n = 3). Significant different letters above vertical bars indicate difference (<i>P</i> <0.05).</p

<i>PmE2F-2</i> mRNA expression profiles after eyestalk ablation.

<p><i>PmE2F-2</i> mRNA relative expression level in ovary tissue post-treatment with eyestalk ablation. Vertical bars represented the mean ± SD (n = 3). Significant different letters above vertical bars indicate difference (P < 0.05).</p

Relative expression levels of <i>PmE2F-2</i> in ovary and hepatopancreas of shrimps after treatment with dsRNA-RBL.

<p>a. Relative expression level of <i>PmE2F-2</i> in the ovary. b. Relative expression level of <i>PmE2F-2</i> in the hepatopancreas. Ovary and hepatopancreas tissues collected from shrimps injected with dsRNA-RBL were compared with respect to <i>PmE2F-2</i> mRNA expression (relative to EF-1α) using Students t-tests. Vertical bars represented mean±SD (n = 3). Significant differences from controls were indicated: **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

Relative expression levels of <i>PmCDK2</i> in ovary and hepatopancreas of shrimps after treatment with dsRNA-E2F-2.

<p>a. Relative expression level of <i>PmCDK2</i> in the ovary. b. Relative expression level of <i>PmCDK2</i> in the hepatopancreas. Vertical bars represented mean±SD (n = 3). Significant differences from controls were indicated: **<i>P</i> < 0.01, *<i>P</i> < 0.05.</p

Genomic structure, expression pattern, and functional characterization of transcription factor E2F-2 from black tiger shrimp (<i>Penaeus monodon</i>)

<div><p>Transcription factor E2F-2 is a regulator of cell cycle. Researchers identified <i>E2F-2</i> genes from yeasts to humans, but few reports investigated <i>E2F-2</i> gene from black tiger shrimp. In the present study, we cloned <i>E2F-2</i> gene from black tiger shrimp (<i>Penaeus monodon</i>). Full-length <i>PmE2F-2</i> complementary DNA sequence measures 3,189 bp with an open reading frame of 1,371 bp. Complete <i>PmE2F-2</i> genomic sequence (17,305 bp) of <i>P</i>. <i>monodon</i> contains nine exons, which are separated by eight introns. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that <i>PmE2F-2</i> is highly expressed in hepatopancreas and ovaries of <i>P</i>. <i>monodon</i>. Highest <i>PmE2F-2</i> expression levels were observed in stage III ovarian development of <i>P</i>. <i>monodon</i>. <i>PmE2F-2</i> expression levels were significantly augmented in ovaries of <i>P</i>. <i>monodon</i> after 5-hydroxytryptamine injection and eyestalk ablation. RNA interference experiments were conducted to examine <i>PmE2F-2</i>, <i>PmCDK2</i>, and <i>PmCyclin E</i> expression profiles. <i>PmE2F-2</i> was successfully knocked down in ovaries and hepatopancreas via double-stranded RNA (dsRNA)–E2F-2 injection. In the same organs, <i>PmE2F-2</i> expression localization and level were investigated through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA—E2F-2 injection, gonadosomatic index of shrimp was significantly lower than those following dsRNA—GFP and phosphate-buffered solution injections. Therefore, <i>PmE2F-2</i> may be involved in ovarian maturation in <i>P</i>. <i>monodon</i>.</p></div

Engineering Antimicrobial Peptides with Improved Antimicrobial and Hemolytic Activities

The rapid rise of antibiotic resistance in pathogens becomes a serious and growing threat to medicine and public health. Naturally occurring antimicrobial peptides (AMPs) are an important line of defense in the immune system against invading bacteria and microbial infection. In this work, we present a combined computational and experimental study of the biological activity and membrane interaction of the computationally designed Bac2A-based peptide library. We used the MARTINI coarse-grained molecular dynamics with adaptive biasing force method and the umbrella sampling technique to investigate the translocation of a total of 91 peptides with different amino acid substitutions through a mixed anionic POPE/POPG (3:1) bilayer and a neutral POPC bilayer, which mimic the bacterial inner membrane and the human red blood cell (hRBC) membrane, respectively. Potential of mean force (PMF, free energy profile) was obtained to measure the free energy barrier required to transfer the peptides from the bulk water phase to the water–membrane interface and to the bilayer interior. Different PMF profiles can indeed identify different membrane insertion scenarios by mapping out peptide–lipid energy landscapes, which are correlated with antimicrobial activity and hemolytic activity. Computationally designed peptides were further tested experimentally for their antimicrobial and hemolytic activities using bacteria growth inhibition assay and hemolysis assay. Comparison of PMF data with cell assay results reveals a good correlation of the peptides between predictive transmembrane activity and antimicrobial/hemolytic activity. Moreover, the most active mutants with the balanced substitutions of positively charged Arg and hydrophobic Trp residues at specific positions were discovered to achieve the improved antimicrobial activity while minimizing red blood cell lysis. Such substitutions provide more effective and cooperative interactions to distinguish the peptide interaction with different lipid bilayers. This work provides a useful computational tool to better understand the mechanism and energetics of membrane insertion of AMPs and to rationally design more effective AMPs

Nucleotide and deduced amino acid sequence of <i>PmE2F-2</i>.

<p>The deduced amino acid sequence is shown below the nucleotide sequence. The initiation code (ATG) and the termination code (TAA) are indicated by the box. The polyadenylation signal sequence (AATAAA) is in bold.</p