CSAR: a contig scaffolding tool using algebraic rearrangements.

Summary: Advances in next generation sequencing have generated massive amounts of short reads. However, assembling genome sequences from short reads still remains a challenging task. Due to errors in reads and large repeats in the genome, many of current assembly tools usually produce just collections of contigs whose relative positions and orientations along the genome being sequenced are still unknown. To address this issue, a scaffolding process to order and orient the contigs of a draft genome is needed for completing the genome sequence. In this work, we propose a new scaffolding tool called CSAR that can efficiently and more accurately order and orient the contigs of a given draft genome based on a reference genome of a related organism. In particular, the reference genome required by CSAR is not necessary to be complete in sequence. Our experimental results on real datasets have shown that CSAR outperforms other similar tools such as Projector2, OSLay and Mauve Aligner in terms of average sensitivity, precision, F-score, genome coverage, NGA50 and running time. Availability and implementation: The program of CSAR can be downloaded from https://github.com/ablab-nthu/CSAR. Contact: hchiu@mail.ncku.edu.tw or cllu@cs.nthu.edu.tw. Supplementary information: Supplementary data are available at Bioinformatics online.

Target-specific identification and characterization of the putative gene cluster for brasilinolide biosynthesis revealing the mechanistic insights and combinatorial synthetic utility of 2-deoxy-l-fucose biosynthetic enzymes.

Brasilinolides exhibiting potent immunosuppressive and antifungal activities with remarkably low toxicity are structurally characterized by an unusual modified 2-deoxy-l-fucose (2dF) attached to a type I polyketide (PK-I) macrolactone. From the pathogenic producer Nocardia terpenica (Nocardia brasiliensis IFM-0406), a 210 kb genomic fragment was identified by target-specific degenerate primers and subsequently sequenced, revealing a giant nbr gene cluster harboring genes (nbrCDEF) required for TDP-2dF biosynthesis and those for PK-I biosynthesis, modification and regulation. The results showed that the genetic and domain arrangements of nbr PK-I synthases agreed colinearly with the PK-I structures of brasilinolides. Subsequent heterologous expression of nbrCDEF in Escherichia coli accomplished in vitro reconstitution of TDP-2dF biosynthesis. The catalytic functions and mechanisms of NbrCDEF enzymes were further characterized by systematic mix-and-match experiments. The enzymes were revealed to display remarkable substrate and partner promiscuity, leading to the establishment of in vitro hybrid deoxysugar biosynthetic pathways throughout an in situ one-pot (iSOP) method. This study represents the first demonstration of TDP-2dF biosynthesis at the enzyme and molecular levels, and provides new hope for expanding the structural diversity of brasilinolides by combinatorial biosynthesis.

CAR: contig assembly of prokaryotic draft genomes using rearrangements.

BACKGROUND: Next generation sequencing technology has allowed efficient production of draft genomes for many organisms of interest. However, most draft genomes are just collections of independent contigs, whose relative positions and orientations along the genome being sequenced are unknown. Although several tools have been developed to order and orient the contigs of draft genomes, more accurate tools are still needed. RESULTS: In this study, we present a novel reference-based contig assembly (or scaffolding) tool, named as CAR, that can efficiently and more accurately order and orient the contigs of a prokaryotic draft genome based on a reference genome of a related organism. Given a set of contigs in multi-FASTA format and a reference genome in FASTA format, CAR can output a list of scaffolds, each of which is a set of ordered and oriented contigs. For validation, we have tested CAR on a real dataset composed of several prokaryotic genomes and also compared its performance with several other reference-based contig assembly tools. Consequently, our experimental results have shown that CAR indeed performs better than all these other reference-based contig assembly tools in terms of sensitivity, precision and genome coverage. CONCLUSIONS: CAR serves as an efficient tool that can more accurately order and orient the contigs of a prokaryotic draft genome based on a reference genome. The web server of CAR is freely available at http://genome.cs.nthu.edu.tw/CAR/ and its stand-alone program can also be downloaded from the same website.

R3D-BLAST: a search tool for similar RNA 3D substructures.

R3D-BLAST is a BLAST-like search tool that allows the user to quickly and accurately search against the PDB for RNA structures sharing similar substructures with a specified query RNA structure. The basic idea behind R3D-BLAST is that all the RNA 3D structures deposited in the PDB are first encoded as 1D structural sequences using a structural alphabet of 23 distinct nucleotide conformations, and BLAST is then applied to these 1D structural sequences to search for those RNA substructures whose 1D structural sequences are similar to that of the query RNA substructure. R3D-BLAST takes as input an RNA 3D structure in the PDB format and outputs all substructures of the hits similar to that of the query with a graphical display to show their structural superposition. In addition, each RNA substructure hit found by R3D-BLAST has an associated E-value to measure its statistical significance. R3D-BLAST is now available online at http://genome.cs.nthu.edu.tw/R3D-BLAST/ for public access.

Faecalibacterium prausnitzii Ameliorates Colorectal Tumorigenesis and Suppresses Proliferation of HCT116 Colorectal Cancer Cells.

Faecalibacterium prausnitzii is one of the most abundant commensals of gut microbiota that is not commonly administered as a probiotic supplement. Being one of the gut's major butyrate-producing bacteria, its clinical significance and uses are on the rise and it has been shown to have anti-inflammatory and gut microbiota-modulating properties in the treatment of inflammatory bowel illness, Crohn's disease, and colorectal cancer. Colorectal cancer (CRC) is a silent killer disease that has become one of the leading causes of cancer-related death worldwide. This study aimed to evaluate the anti-tumorigenic and antiproliferative role of F. prausnitzii as well as to study its effects on the diversity of gut microbiota in rats. Findings showed that F. prausnitzii probiotic significantly reduced the colonic aberrant crypt foci frequency and formation in Azoxymethane (AOM)-induced CRC in rats. In addition, the administration of F. prausnitzii lowered the lipid peroxidation levels in the colon tissues. For in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, the cell-free supernatant of F. prausnitzii suppressed the growth of HCT116 colorectal cancer cells in a time/dose-dependent manner. 16S rRNA gene sequencing using rat stool samples showed that the administration of F. prausnitzii modulated the gut microbiota of the rats and enhanced its diversity. Hence, these findings suggest that F. prausnitzii as a probiotic supplement can be used in CRC prevention and management; however, more studies are warranted to understand its cellular and molecular mechanisms of action.

Reconstructing genome trees of prokaryotes using overlapping genes.

BACKGROUND: Overlapping genes (OGs) are defined as adjacent genes whose coding sequences overlap partially or entirely. In fact, they are ubiquitous in microbial genomes and more conserved between species than non-overlapping genes. Based on this property, we have previously implemented a web server, named OGtree, that allows the user to reconstruct genome trees of some prokaryotes according to their pairwise OG distances. By analogy to the analyses of gene content and gene order, the OG distance between two genomes we defined was based on a measure of combining OG content (i.e., the normalized number of shared orthologous OG pairs) and OG order (i.e., the normalized OG breakpoint distance) in their whole genomes. A shortcoming of using the concept of breakpoints to define the OG distance is its inability to analyze the OG distance of multi-chromosomal genomes. In addition, the amount of overlapping coding sequences between some distantly related prokaryotic genomes may be limited so that it is hard to find enough OGs to properly evaluate their pairwise OG distances. RESULTS: In this study, we therefore define a new OG order distance that is based on more biologically accurate rearrangements (e.g., reversals, transpositions and translocations) rather than breakpoints and that is applicable to both uni-chromosomal and multi-chromosomal genomes. In addition, we expand the term "gene" to include both its coding sequence and regulatory regions so that two adjacent genes whose coding sequences or regulatory regions overlap with each other are considered as a pair of overlapping genes. This is because overlapping of regulatory regions of distinct genes suggests that the regulation of expression for these genes should be more or less interrelated. Based on these modifications, we have reimplemented our OGtree as a new web server, named OGtree2, and have also evaluated its accuracy of genome tree reconstruction on a testing dataset consisting of 21 Proteobacteria genomes. Our experimental results have finally shown that our current OGtree2 indeed outperforms its previous version OGtree, as well as another similar server, called BPhyOG, significantly in the quality of genome tree reconstruction, because the phylogenetic tree obtained by OGtree2 is greatly congruent with the reference tree that coincides with the taxonomy accepted by biologists for these Proteobacteria. CONCLUSIONS: In this study, we have introduced a new web server OGtree2 at http://bioalgorithm.life.nctu.edu.tw/OGtree2.0/ that can serve as a useful tool for reconstructing more precise and robust genome trees of prokaryotes according to their overlapping genes.

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation.

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi-quantitative RT-PCR indicated that PEMFs significantly altered temporal expression of osteogenesis-related genes, including a 2.7-fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC-mediated osteogenesis, and accelerate the osteogenesis.

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells.

Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF-exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm2, respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20-60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF-exposed BMMSC showed multi-lineage differentiation potential similar to the control group.

Analysis of circular genome rearrangement by fusions, fissions and block-interchanges.

BACKGROUND: Analysis of genomes evolving via block-interchange events leads to a combinatorial problem of sorting by block-interchanges, which has been studied recently to evaluate the evolutionary relationship in distance between two biological species since block-interchange can be considered as a generalization of transposition. However, for genomes consisting of multiple chromosomes, their evolutionary history should also include events of chromosome fusions and fissions, where fusion merges two chromosomes into one and fission splits a chromosome into two. RESULTS: In this paper, we study the problem of genome rearrangement between two genomes of circular and multiple chromosomes by considering fusion, fission and block-interchange events altogether. By use of permutation groups in algebra, we propose an O (n2) time algorithm to efficiently compute and obtain a minimum series of fusions, fissions and block-interchanges required to transform one circular multi-chromosomal genome into another, where n is the number of genes shared by the two studied genomes. In addition, we have implemented this algorithm as a web server, called FFBI, and have also applied it to analyzing by gene orders the whole genomes of three human Vibrio pathogens, each with multiple and circular chromosomes, to infer their evolutionary relationships. Consequently, our experimental results coincide well with our previous results obtained using the chromosome-by-chromosome comparisons by landmark orders between any two Vibrio chromosomal sequences as well as using the traditional comparative analysis of 16S rRNA sequences. ConclusionFFBI is a useful tool for the bioinformatics analysis of circular and multiple genome rearrangement by fusions, fissions and block-interchanges.

Functional characterization and substrate specificity of spinosyn rhamnosyltransferase by in vitro reconstitution of spinosyn biosynthetic enzymes.

Spinosyn, a potent insecticide, is a novel tetracyclic polyketide decorated with d-forosamine and tri-O-methyl-L-rhamnose. Spinosyn rhamnosyltransferase (SpnG) is a key biocatalyst with unique sequence identity and controls the biosynthetic maturation of spinosyn. The rhamnose is critical for the spinosyn insecticidal activity and cell wall biosynthesis of the spinosyn producer, Saccharopolyspora spinosa. In this study, we have functionally expressed and characterized SpnG and the three enzymes, Gdh, Epi, and Kre, responsible for dTDP-L-rhamnose biosynthesis in S. spinosa by purified enzymes from Escherichia coli. Most notably, the substrate specificity of SpnG was thoroughly characterized by kinetic and inhibition experiments using various NDP sugar analogs made by an in situ combination of NDP-sugar-modifying enzymes. SpnG was found to exhibit striking substrate promiscuity, yielding corresponding glycosylated variants. Moreover, the critical residues presumably involved in catalytic mechanism of Gdh and SpnG were functionally evaluated by site-directed mutagenesis. The information gained from this study has provided important insight into molecular recognition and mechanism of the enzymes, especially SpnG. The results have made possible the structure-activity characterization of SpnG, as well as the use of SpnG or its engineered form to serve as a combinatorial tool to make spinosyn analogs with altered biological activities and potency.

Molecular cloning, sequence analysis and functional characterization of the gene cluster for biosynthesis of K-252a and its analogs.

Among the indolocarbazole alkaloids of antitumor antibiotics, K-252a represents a structurally unique indolocarbazole glycoside and exhibits potent neuroprotective and broad anticancer activities. K-252a consists of K-252c and the unusual dihydrostreptose moiety, linked together with oxidative and glycosidic C-N bonds. Herein, we reported a complete sequence of an approximately 45 kb genomic fragment harboring the gene cluster for the biosynthesis of indolocarbazole alkaloids in Nocardiopsis sp. K-252 (NRRL15532). The sequence of 35 open reading frames discovered several new, critical genes, hence shedding new light on biosynthesis, resistance and regulation of K-252a and its analogs. To functionally characterize the gene cluster in vitro and in enzyme level, a multigene expression cassette containing the K-252c biosynthetic genes was constructed and successfully overexpressed in Escherichia coli to yield soluble proteins for cell-free tandem enzymatic assays. Consequently, the heterologous expression with soluble NokA and NokB led to in vitro production of chromopyrrolic acid (CPA), thereby providing functional evidence for K-252c biosynthesis. Moreover, a facile production of CPA in culture broth was successfully accomplished by using an in vivo biotransformation of L-tryptophan with E. coli harboring the gene cassette. Importantly, by sequence analysis and the functional characterization here and in the companion paper, biosynthetic pathways leading to formation of K-252a and its analogs were hence proposed. Together, the results provide critical information and materials useful for combinatorial biosynthesis of K-252a and its analogs for therapeutic applications.

Biochemical characterization and substrate specificity of the gene cluster for biosyntheses of K-252a and its analogs by in vitro heterologous expression system of Escherichia coli.

The indolocarbazole family of natural products has attracted great attention because of their unique structural features and potential therapeutic applications. Structurally distinct in the family, K-252a is characterized by an unusual dihydrostreptose moiety cross-bridged to K-252c aglycone with two C-N linkages. K-252a has served as a valuable lead for treatments of various cancers and neurodegenerative disorders. Recent cloning of the nok gene cluster for biosyntheses of K-252a and its analogs from Nocardiopsis sp. K-252 (NRRL15532) has revealed the nokABCD genes indispensible for K-252c biosynthesis and the key gene (nokL) coding for N-glycosylation. Herein, we report the first, successful demonstration of in vitro sugar transferase activity of indolocarbazole N-glycosyltransferase (NokL) by use of soluble protein expressed from Escherichia coli. Notably, NokL was found to exhibit peculiar mode of substrate promiscuity. Moreover, NokA and NokB reactions were biochemically characterized thoroughly by natural and alternative (e.g. fluoro-) substrates and by ammonium hydroxide (NH(4)OH). Interestingly, the in vitro expression of NokA revealed high substrate stereoselectivity, giving several indole-3-pyruvic acid-derived compounds, including indol-3-carboxaldehyde (ICA) and indole-3-acetic acid. The use of NH(4)OH successfully dissected the in vitro NokA/NokB coupled reaction, revealing mechanistic insight into the enzymes and their cross-talking relationship. Also, a simple, useful method to synthesize K-252d, ICA and chromopyrrolic acid (the NokB product) was developed by the E. coli expression systems of NokL, NokA and NokA/NokB, respectively. Together with NokA and NokB, NokL may serve as a useful tool for combinatorial engineering of K-252a and its analogs for improved therapeutic values.

A heuristic approach for detecting RNA H-type pseudoknots.

MOTIVATION: RNA H-type pseudoknots are ubiquitous pseudoknots that are found in almost all classes of RNA and thought to play very important roles in a variety of biological processes. Detection of these RNA H-type pseudoknots can improve our understanding of RNA structures and their associated functions. However, the currently existing programs for detecting such RNA H-type pseudoknots are still time consuming and sometimes even ineffective. Therefore, efficient and effective tools for detecting the RNA H-type pseudoknots are needed. RESULTS: In this paper, we have adopted a heuristic approach to develop a novel tool, called HPknotter, for efficiently and accurately detecting H-type pseudoknots in an RNA sequence. In addition, we have demonstrated the applicability and effectiveness of HPknotter by testing on some sequences with known H-type pseudoknots. Our approach can be easily extended and applied to other classes of more general pseudoknots. AVAILABILITY: The web server of our HPknotter is available for online analysis at http://bioalgorithm.life.nctu.edu.tw/HPKNOTTER/ CONTACT: cllu@mail.nctu.edu.tw, chiu@cc.nctu.edu.tw