Monoclonal Antibodies against Accumulation-Associated Protein Affect EPS Biosynthesis and Enhance Bacterial Accumulation of Staphylococcus epidermidis

Because there is no effective antibiotic to eradicate Staphylococcus epidermidis biofilm infections that lead to the failure of medical device implantations, the development of anti-biofilm vaccines is necessary. Biofilm formation by S. epidermidis requires accumulation-associated protein (Aap) that contains sequence repeats known as G5 domains, which are responsible for the Zn2+-dependent dimerization of Aap to mediate intercellular adhesion. Antibodies against Aap have been reported to inhibit biofilm accumulation. In the present study, three monoclonal antibodies (MAbs) against the Aap C-terminal single B-repeat construct followed by the 79-aa half repeat (AapBrpt1.5) were generated. MAb18B6 inhibited biofilm formation by S. epidermidis RP62A to 60% of the maximum, while MAb25C11 and MAb20B9 enhanced biofilm accumulation. All three MAbs aggregated the planktonic bacteria to form visible cell clusters. Epitope mapping revealed that the epitope of MAb18B6, which recognizes an identical area within AapBrpt constructs from S. epidermidis RP62A, was not shared by MAb25C11 and MAb20B9. Furthermore, all three MAbs were found to affect both Aap expression and extracellular polymeric substance (EPS, including extracellular DNA and PIA) biosynthesis in S. epidermidis and enhance the cell accumulation. These findings contribute to a better understanding of staphylococcal biofilm formation and will help to develop epitope-peptide vaccines against staphylococcal infections

Novel electro-hydraulic position control system for primary mirror supporting system

In the field of modern large-scale telescope, primary mirror supporting system technology faces the difficulties of theoretically uniform output force request and bias compensation. Therefore, a novel position control system combining hydraulic system with servo motor system is introduced. The novel system ensures uniform output force on supporting points without complicating the mechanical structure. The structures of both primary mirror supporting system and novel position system are described. Then, the mathematical model of novel position control system is derived for controller selection. A proportional–derivative controller is adopted for simulations and experiments of step response and triangle path tracking. The results show that proportional–derivative controller guarantees the system with micrometer-level positioning ability. A modified proportional–derivative controller is utilized to promote system behavior with faster response overshoot. The novel position control system is then applied on primary mirror supporting system. Coupling effect is observed among actuator partitions, and relocation of virtual pivot supporting point is chosen as the decoupling measurement. The position keeping ability of the primary mirror supporting system is verified by rotating the mirror cell at a considerably high rate. The experiment results show that the decoupled system performs better with smaller bias and shorter recovery time

Design, Synthesis and Anticancer Evaluation of Fangchinoline Derivatives

Twenty fangchinoline derivatives were synthesized from the natural product fangchinoline, and their anticancer activities on human breast cancer MDA-MB-231 cell line, human prostate cancer PC3 cell line, human melanoma WM9 cell line and human leukaemia HEL and K562 cell lines were evaluated. The biological result showed that those derivatives exhibited potent activities on inhibiting cancer cell growth, and the structure-activity relationships were investigated. Among them, compound 4g, which was protected by benzoyl group in 7-phenolic position and nitrified in 14-position, showed impressive inhibition on all 5 cancer cell lines, especially WM9 cell line, with an IC50 value of 1.07 µM. Further mechanistic studies demonstrated that compound 4g may induce cancer cell death by apoptotic means. These research results suggested that compound 4g could be a lead for the further development toward an anticancer agent against human melanoma WM9 in the future

Vulgarisin A, a New Diterpenoid with a Rare 5/6/4/5 Ring Skeleton from the Chinese Medicinal Plant <i>Prunella vulgaris</i>

Vulgarisin A (<b>1</b>), a new diterpenoid with an unprecedented 5/6/4/5 fused tetracyclic ring skeleton, has been isolated from the medicinal plant <i>Prunella vulgaris</i> Linn. Its structure was characterized by extensive spectroscopic methods, and the absolute configuration was secured by single crystal X-ray diffraction analysis. Compound <b>1</b> showed weak cytotoxicity against human lung carcinoma A549 cells with an IC₅₀ value of 57.0 μM

Aap expression in biofilms of <i>S. epidermidis</i>.

<p>Aap in the biofilms of <i>S. epidermidis</i> RP62A was probed with MAb_25C11 (10 ng/mL) and Cy3-conjugated secondary antibody (1∶100 diluted, red fluorescence), and the bacteria were further stained with SYTO9 (1 µM, green fluorescence). Aap expression was observed under a Leica TCS SP5 CLSM. Confocal microscopy Z-series of the biofilms were acquired in 0.5-µm increments. “PC”: positive control (antigens contained in the biofilm were probed using mouse anti-<i>S. epidermidis</i> serum (1∶400 diluted) and Cy3-conjugated secondary antibody, showing that antibodies could diffuse to the inner side of the biofilm), “NC”: negative control (the biofilm formed in the presence of MAb_25C11 was probed with Cy3-conjugated secondary antibody alone to establish that the MAb-treated biofilms no longer contained initially added MAb (10 µg/mL), that could cause false-positive immunofluorescence, after 14 h culture), “RP62A”: untreated, “Mock”: normal mouse IgG-treated, the red arrow indicates the crater-like micropores.</p

Oligonucleotide primers used in the present study.

<p><b>a</b>. Primers were designed according to the genomic sequence of <i>S. epidermidis</i> ATCC 12228 (GenBank NC_004461).</p><p><b>b</b>. Primers were designed according to the genomic sequence of <i>S. epidermidis</i> RP62A (GenBank NC_002976).</p><p><b>c</b>. Primers were designed according to the gene sequence of the 56-residue B1 immunoglobulin binding domain (GB1) of immunoglobulin G-binding protein from <i>Streptococcus dysgalactiae</i> subsp. <i>equisimilis</i> GGS_124 (amino acids 303–357, GenBank YP_002997067).</p

Fluorescence quantities of the Live/Dead stained biofilms.^a

<p><b>a</b>. The biofilm of <i>S. epidermidis</i> RP62A formed in the presence of each MAb (10 µg/mL) was visualized using Live/Dead viability staining (SYTO9/PI). After obtaining the 3-D structure of the biofilms under a CLSM (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020918#pone-0020918-g003" target="_blank">Figure 3</a>), the Z-stack composite confocal photomicrographs were further generated, and the stacks of viable cells, dead cells, and both cells (viable & dead) were generated separately. The fluorescence quantities of SYTO9-stained viable cells and PI-stained dead cells were determined using ImageJ program (<a href="http://rsbweb.nih.gov/ij" target="_blank">http://rsbweb.nih.gov/ij</a>).</p><p><b>b</b>. “SYTO9” stands for the fluorescence quantities of the viable cell stacks.</p><p><b>c</b>. “PI” stands for the fluorescence quantities of the dead cell stacks.</p><p><b>d</b>. “Total” represents the fluorescence quantities of both cell stacks.</p><p><b>e</b>. “PI/Total” represents the proportion of the dead cell in the biofilms.</p