A Novel Anti-Tumor Inhibitor Identified by Virtual Screen with PLK1 Structure and Zebrafish Assay

Polo-like kinase 1 (PLK1), one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC₅₀ values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.     

A New Molecular Phylogeny and a New Genus,Pendulorchis, of the Aerides–Vanda Alliance (Orchidaceae: Epidendroideae)

Background

The Aerides–Vanda alliance is a complex group in the subtribe Aeridinae (subfamily Epidendroideae, Orchidaceae). Some phylogenetic systems of this alliance have been previously proposed based on molecular and morphological analyses. However, several taxonomic problems within this alliance as well as between it and its allies remain unsolved.

Methodology/Principal Findings

We utilized ITS and five plastid DNA regions in this phylogenetic analysis. Consensus trees strongly indicate that the Aerides–Vanda alliance is monophyletic, and the 14 genera of this alliance can be grouped into the following clades with 14 subclades: 1. Aerides, comprising two subclades: Rhynchostylis and Aerides; 2. Ascocentropsis; 3. Papilionanthe; 4. Vanda, comprising five subclades: Neofinetia, Christensonia, Seidenfadenia, Ascocentrum, and Vanda–Trudelia, in which Vanda and Trudelia form a subclade; 5. Tsiorchis, comprising three subclades: Chenorchis, Tsiorchis, and two species of Ascocentrum; 6. Paraholcoglossum; and 7. Holcoglossum. Among the 14 genera, only Ascocentrum is triphyletic: two species of the Ascocentrum subclade, an independent subclade Ascocentrum subclade in the Tsiorchis clade; the Ascocentrum subclade in the Vanda clade; and one species in the Holcoglossum clade. The Vanda and Trudelia species belong to the same subclade. The molecular conclusion is consistent with their morphological characteristics.

Conclusions

We elucidate the relationship among the 14 genera of the Aerides–Vanda alliance. Our phylogenetic results reveal that the Aerides–Vanda alliance is monophyletic, but it can be divided into 14 genera. The data prove that Ascocentrum is triphyletic. Plants with elongate-terete leaves and small flowers should be treated as a new genus, Pendulorchis. Saccolabium himalaicum (Ascocentrum himalaicum) should be transferred to Pendulorchis. Ascocentrum pumilum, endemic to Taiwan, should be transferred to Holcoglossum. A new combination, Holcoglossum pumilum, was also established. Trudelia should not be recognized as an independent genus. Two new species, Pendulorchis gaoligongensis and Holcoglossum singchianum, were described as well.     

A Hypomorphic Lsd1 Allele Results in Heart Development Defects in Mice

Lysine-specific demethylase 1 (Lsd1/Aof2/Kdm1a), the first enzyme with specific lysine demethylase activity to be described, demethylates histone and non-histone proteins and is essential for mouse embryogenesis. Lsd1 interacts with numerous proteins through several different domains, most notably the tower domain, an extended helical structure that protrudes from the core of the protein. While there is evidence that Lsd1-interacting proteins regulate the activity and specificity of Lsd1, the significance and roles of such interactions in developmental processes remain largely unknown. Here we describe a hypomorphic Lsd1 allele that contains two point mutations in the tower domain, resulting in a protein with reduced interaction with known binding partners and decreased enzymatic activity. Mice homozygous for this allele die perinatally due to heart defects, with the majority of animals suffering from ventricular septal defects. Molecular analyses revealed hyperphosphorylation of E-cadherin in the hearts of mutant animals. These results identify a previously unknown role for Lsd1 in heart development, perhaps partly through the control of E-cadherin phosphorylation.     

Study of Double-Side Ultrasonic Embossing for Fabrication of Microstructures on Thermoplastic Polymer Substrates

Double-side replication of polymer substrates is beneficial to the design and the fabrication of 3-demensional devices. The ultrasonic embossing method is a promising, high efficiency and low cost replication method for thermoplastic substrates. It is convenient to apply silicon molds in ultrasonic embossing, because microstructures can be easily fabricated on silicon wafers with etching techniques. To reduce the risk of damaging to silicon molds and to improve the replication uniformity on both sides of the polymer substrates, thermal assisted ultrasonic embossing method was proposed and tested. The processing parameters for the replication of polymethyl methacrylate (PMMA), including ultrasonic amplitude, ultrasonic force, ultrasonic time, and thermal assisted temperature were studied using orthogonal array experiments. The influences of the substrate thickness, pattern style and density were also investigated. The experiment results show that the principal parameters for the upper and lower surface replication are ultrasonic amplitude and thermal assisted temperature, respectively. As to the replication uniformity on both sides, the ultrasonic force has the maximal influence. Using the optimized parameters, the replication rate reached 97.5% on both sides of the PMMA substrate, and the cycle time was less than 50 s.     

Higher Isolation of NDM-1 Producing Acinetobacter baumannii from the Sewage of the Hospitals in Beijing

Multidrug resistant microbes present in the environment are a potential public health risk. In this study, we investigate the presence of New Delhi metallo-β-lactamase 1 (NDM-1) producing bacteria in the 99 water samples in Beijing City, including river water, treated drinking water, raw water samples from the pools and sewage from 4 comprehensive hospitals. For the bla _NDM-1 positive isolate, antimicrobial susceptibility testing was further analyzed, and Pulsed Field Gel Electrophoresis (PFGE) was performed to determine the genetic relationship among the NDM-1 producing isolates from sewage and human, as well as the clinical strains without NDM-1. The results indicate that there was a higher isolation of NDM-1 producing Acinetobacter baumannii from the sewage of the hospitals, while no NDM-1 producing isolates were recovered from samples obtained from the river, drinking, or fishpond water. Surprisingly, these isolates were markedly different from the clinical isolates in drug resistance and pulsed field gel electrophoresis profiles, suggesting different evolutionary relationships. Our results showed that the hospital sewage may be one of the diffusion reservoirs of NDM-1 producing bacteria.     

Automatic Peak Selection by a Benjamini-Hochberg-Based Algorithm

A common issue in bioinformatics is that computational methods often generate a large number of predictions sorted according to certain confidence scores. A key problem is then determining how many predictions must be selected to include most of the true predictions while maintaining reasonably high precision. In nuclear magnetic resonance (NMR)-based protein structure determination, for instance, computational peak picking methods are becoming more and more common, although expert-knowledge remains the method of choice to determine how many peaks among thousands of candidate peaks should be taken into consideration to capture the true peaks. Here, we propose a Benjamini-Hochberg (B-H)-based approach that automatically selects the number of peaks. We formulate the peak selection problem as a multiple testing problem. Given a candidate peak list sorted by either volumes or intensities, we first convert the peaks into -values and then apply the B-H-based algorithm to automatically select the number of peaks. The proposed approach is tested on the state-of-the-art peak picking methods, including WaVPeak [1] and PICKY [2]. Compared with the traditional fixed number-based approach, our approach returns significantly more true peaks. For instance, by combining WaVPeak or PICKY with the proposed method, the missing peak rates are on average reduced by 20% and 26%, respectively, in a benchmark set of 32 spectra extracted from eight proteins. The consensus of the B-H-selected peaks from both WaVPeak and PICKY achieves 88% recall and 83% precision, which significantly outperforms each individual method and the consensus method without using the B-H algorithm. The proposed method can be used as a standard procedure for any peak picking method and straightforwardly applied to some other prediction selection problems in bioinformatics. The source code, documentation and example data of the proposed method is available at http://sfb.kaust.edu.sa/pages/software.aspx.     

Benchmarking Human Protein Complexes to Investigate Drug-Related Systems and Evaluate Predicted Protein Complexes

Protein complexes are key entities to perform cellular functions. Human diseases are also revealed to associate with some specific human protein complexes. In fact, human protein complexes are widely used for protein function annotation, inference of human protein interactome, disease gene prediction, and so on. Therefore, it is highly desired to build an up-to-date catalogue of human complexes to support the research in these applications. Protein complexes from different databases are as expected to be highly redundant. In this paper, we designed a set of concise operations to compile these redundant human complexes and built a comprehensive catalogue called CHPC2012 (Catalogue of Human Protein Complexes). CHPC2012 achieves a higher coverage for proteins and protein complexes than those individual databases. It is also verified to be a set of complexes with high quality as its co-complex protein associations have a high overlap with protein-protein interactions (PPI) in various existing PPI databases. We demonstrated two distinct applications of CHPC2012, that is, investigating the relationship between protein complexes and drug-related systems and evaluating the quality of predicted protein complexes. In particular, CHPC2012 provides more insights into drug development. For instance, proteins involved in multiple complexes (the overlapping proteins) are potential drug targets; the drug-complex network is utilized to investigate multi-target drugs and drug-drug interactions; and the disease-specific complex-drug networks will provide new clues for drug repositioning. With this up-to-date reference set of human protein complexes, we believe that the CHPC2012 catalogue is able to enhance the studies for protein interactions, protein functions, human diseases, drugs, and related fields of research. CHPC2012 complexes can be downloaded from http://www1.i2r.a-star.edu.sg/xlli/CHPC2012/CHPC2012.htm.     

Molecular Analysis of Anaplasma phagocytophilum Isolated from Patients with Febrile Diseases of Unknown Etiology in China

Although anaplasmosis cases have been nationally identified in China, no human isolates of A. phagocytophilum have been obtained, which limits the analysis of any molecular and genetic contributions to patients'' severe clinical manifestations and the study of the bacteria''s pathogeneses in China. Given this situation, a joint project was conducted in 2009–2010. A total of 421 febrile cases of unknown etiology were collected and the patients'' blood samples were collected for laboratory diagnoses including serologic diagnosis based on the four-fold rise in the anti- A. phagocytophilum IgG titer by indirect micro-immunofluorescence assay (IFA), positive PCR assay and confirmation of A. phagocytophilum DNA and positive culture of A. phagocytophilum and confirmed by amplification and sequencing of the 16S rRNA and ank A genes of the A. phagocytophilum isolates. A total of 570 ticks were collected from the patients'' domestic animals (456) and from wild fields (114) for culturing and amplifying and sequencing the 16S rRNA gene of A. phagocytophilum. Phylogenetic analyses were performed on the 16S rRNA and ank A gene sequences of the isolates and the ticks tested in the study. A total of 46 (10.9%) confirmed and 16 (3.8%) probable cases were diagnosed and severe clinical features and higher mortality rates were observed in these Chinese patients. Five isolates were obtained and the 16S rRNA genes of the 5 isolates were conserved but variety for ank A genes. Two human isolates and 1 tick isolate from Shandong Peninsula, where all patients exhibited severe clinical manifestations, were grouped as one clan based on the phylogenetic analyses, while 2 other human isolates were clustered in a second clan. 43.5% of H. longicornis were infected with A. phagocytophilum.The present study is the first to obtain clinical isolates of A. phagocytophilum in China. The diversity of the ank A genes of Chinese isolates will help us to further discern the relationship between the variations in the ank A genes and the severity of the disease''s clinical manifestations in China.