Split-inducing indels in phylogenomic analysis

Background

Most phylogenetic studies using molecular data treat gaps in multiple sequence alignments as missing data or even completely exclude alignment columns that contain gaps.

Results

Here we show that gap patterns in large-scale, genome-wide alignments are themselves phylogenetically informative and can be used to infer reliable phylogenies provided the gap data are properly filtered to reduce noise introduced by the alignment method. We introduce here the notion of split-inducing indels (splids) that define an approximate bipartition of the taxon set. We show both in simulated data and in case studies on real-life data that splids can be efficiently extracted from phylogenomic data sets.

Conclusions

Suitably processed gap patterns extracted from genome-wide alignment provide a surprisingly clear phylogenetic signal and an allow the inference of accurate phylogenetic trees.

     

Iridoid and phenylethanoid/phenylpropanoid metabolite profiles of <Emphasis Type="Italic">Scrophularia</Emphasis> and <Emphasis Type="Italic">Verbascum</Emphasis> species used medicinally in North America

Introduction

Botanicals containing iridoid and phenylethanoid/phenylpropanoid glycosides are used worldwide for the treatment of inflammatory musculoskeletal conditions that are primary causes of human years lived with disability, such as arthritis and lower back pain.

Objectives

We report the analysis of candidate anti-inflammatory metabolites of several endemic Scrophularia species and Verbascum thapsus used medicinally by peoples of North America.

Methods

Leaves, stems, and roots were analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and partial least squares-discriminant analysis (PLS-DA) was performed in MetaboAnalyst 3.0 after processing the datasets in Progenesis QI.

Results

Comparison of the datasets revealed significant and differential accumulation of iridoid and phenylethanoid/phenylpropanoid glycosides in the tissues of the endemic Scrophularia species and Verbascum thapsus.

Conclusions

Our investigation identified several species of pharmacological interest as good sources for harpagoside and other important anti-inflammatory metabolites.

     

Extracting DNA words based on the sequence features: non-uniform distribution and integrity

Background

DNA sequence can be viewed as an unknown language with words as its functional units. Given that most sequence alignment algorithms such as the motif discovery algorithms depend on the quality of background information about sequences, it is necessary to develop an ab initio algorithm for extracting the “words” based only on the DNA sequences.

Methods

We considered that non-uniform distribution and integrity were two important features of a word, based on which we developed an ab initio algorithm to extract “DNA words” that have potential functional meaning. A Kolmogorov-Smirnov test was used for consistency test of uniform distribution of DNA sequences, and the integrity was judged by the sequence and position alignment. Two random base sequences were adopted as negative control, and an English book was used as positive control to verify our algorithm. We applied our algorithm to the genomes of Saccharomyces cerevisiae and 10 strains of Escherichia coli to show the utility of the methods.

Results

The results provide strong evidences that the algorithm is a promising tool for ab initio building a DNA dictionary.

Conclusions

Our method provides a fast way for large scale screening of important DNA elements and offers potential insights into the understanding of a genome.

     

Comparative analysis of the mitochondrial genomes of oriental spittlebug trible Cosmoscartini: insights into the relationships among closely related taxa

Background

Cosmoscartini (Hemiptera: Cercopoidea: Cercopidae) is a large and brightly colored Old World tropical tribe, currently containing over 310 phytophagous species (including some economically important pests of eucalyptus in China) in approximately 17 genera. However, very limited information of Cosmoscartini is available except for some scattered taxonomic studies. Even less is known about its phylogenetic relationship, especially among closely related genera or species. In this study, the detailed comparative genomic and phylogenetic analyses were performed on nine newly sequenced mitochondrial genomes (mitogenomes) of Cosmoscartini, with the purpose of exploring the taxonomic status of the previously defined genus Okiscarta and some closely related species within the genus Cosmoscarta.

Results

Mitogenomes of Cosmoscartini display similar genomic characters in terms of gene arrangement, nucleotide composition, codon usage and overlapping regions. However, there are also many differences in intergenic spacers, mismatches of tRNAs, and the control region. Additionally, the secondary structures of rRNAs within Cercopidae are inferred for the first time.Based on comparative genomic (especially for the substitution pattern of tRNA secondary structure) and phylogenetic analyses, the representative species of Okiscarta uchidae possesses similar structures with other Cosmoscarta species and is placed consistently in Cosmoscarta. Although Cosmoscarta bimacula is difficult to be distinguished from Cosmoscarta bispecularis by traditional morphological methods, evidence from mitogenomes highly support the relationships of (C. bimacula?+?Cosmoscarta rubroscutellata)?+?(C. bispecularis?+?Cosmoscarta sp.).

Conclusions

This study presents mitogenomes of nine Cosmoscartini species and represents the first detailed comparative genomic and phylogenetic analyses within Cercopidae. It is indicated that knowledge of mitogenomes can be effectively used to resolve phylogenetic relationships at low taxonomic levels. Sequencing more mitogenomes at various taxonomic levels will also improve our understanding of mitogenomic evolution and phylogeny in Cercopidae.

     

A new method for alignment of LC-MALDI-TOF data

Background

In proteomics studies, liquid chromatography coupled to mass spectrometry (LC-MS) has proven to be a powerful technology to investigate differential expression of proteins/peptides that are characterized by their peak intensities, mass-to-charge ratio (m/z), and retention time (RT). The variable complexity of peptide mixtures and occasional drifts lead to substantial variations in m/z and RT dimensions. Thus, label-free differential protein expression studies by LC-MS technology require alignment with respect to both RT and m/z to ensure that same proteins/peptides are compared from multiple runs.

Methods

In this study, we propose a new strategy to align LC-MALDI-TOF data by combining quality threshold cluster analysis and support vector regression. Our method performs alignment on the basis of measurements in three dimensions (RT, m/z, intensity).

Results and conclusions

We demonstrate the suitability of our proposed method for alignment of LC-MALDI-TOF data through a previously published spike-in dataset and a new in-house generated spike-in dataset. A comparison of our method with other methods that utilize only RT and m/z dimensions reveals that the use of intensity measurements enhances alignment performance.

     

Retrieving hidden atrial repolarization waves from standard surface ECGs

Background

This study estimates atrial repolarization activities (Ta waves), which are typically hidden most of the time from body surface electrocardiography when diagnosing cardiovascular diseases. The morphology of Ta waves has been proven to be an important marker for the early sign of inferior injury, such as acute atrial infarction, or arrhythmia, such as atrial fibrillation. However, Ta waves are usually unseen except during conduction system malfunction, such as long QT interval or atrioventricular block. Therefore, justifying heart diseases based on atrial repolarization becomes impossible in sinus rhythm.

Methods

We obtain TMPs in the atrial part of the myocardium which reflects the correct excitation sequence starting from the atrium to the end of the apex.

Results

The resulting TMP shows the hidden atrial part of ECG waves.

Conclusions

This extraction makes many diseases, such as acute atrial infarction or arrhythmia, become easily diagnosed.

     

Functional characterization and substrate specificity analysis of Δ6-desaturase from marine microalga <Emphasis Type="Italic">Isochrysis</Emphasis> sp.

Objectives

To express a Δ6-desaturase gene and produce gamma-linolenic acid (GLA) and stearidonic acid (SDA) in prokaryotic expression system (Escherichia coli), and analyze its substrate specificity in the omega-3 fatty acid biosynthetic pathway.

Results

Full-length ORF (1448 bp) of Δ6Des-Iso was isolated from Isochrysis sp. and characterized using multiple sequence alignment, phylogenetic analysis, transmembrane domain, and protein tertiary structure. Δ6Des-Iso is a front-end desaturase consisting of three conserved histidine domains and a cytochrome b₅ domain. Δ6Des-Iso was cloned and expressed in E. coli with the production of GLA and SDA. Recombinant E. coli utilized 27 and 8% of exogenously supplied alpha-linolenic acid (ALA) and linoleic acid (LA) to produce 6.3% of SDA and 2.3% of GLA, respectively, suggesting that isolated Δ6Des-Iso is specific to the omega-3 pathway.

Conclusion

For the first time production of GLA and SDA in a prokaryotic system was achieved.

     

Identification of an oleosin-like gene in seagrass seeds

Objective

To investigate the oil body protein and function in seeds of mature seagrass, Thalassia hemprichii.

Results

Seeds of mature seagrass T. hemprichii when stained with a fluorescent probe BODIPY showed the presence of oil bodies in intracellular cells. Triacylglycerol was the major lipid class in the seeds. Protein extracted from seagrass seeds was subjected to immunological cross-recognition with land plant seed oil body proteins, such as oleosin and caleosin, resulting in no cross-reactivity. An oleosin-like gene was found in seagrass seeds. Next generation sequencing and sequence alignment indicated that the deduced seagrass seed oleosin-like protein has a central hydrophobic domain responsible for their anchoring onto the surface of oil bodies. Phylogenetic analysis showed that the oleosin-like protein was evolutionarily closer to pollen oleosin than to seed oleosins.

Conclusion

Oil body protein found in seagrass seeds represent a distinct class of land seed oil body proteins.

     

Species of family <Emphasis Type="Italic">Promicromonosporaceae</Emphasis> and family <Emphasis Type="Italic">Cellulomonadeceae</Emphasis> that produce cellulosome-like multiprotein complexes

Objectives

To screen the phylogenetically-nearest members of Cellulosimicrobium cellulans for the production of cellulosome-like multienzyme complexes and extracellular β-xylosidase activity against 7-xylosyltaxanes and to get corresponding molecular insights.

Results

Cellulosimicrobium (family Promicromonosporaceae) and all genera of the family Cellulomonadeceaec produced both cellulosome-like multienzyme complexes and extracellular β-xylosidase activity, while the other genera of the family Promicromonosporaceae did not. Multiple sequence alignments further indicated that hypothetic protein M768_06655 might be a possible key subunit.

Conclusion

This is the first report that many actinobacteria species can produce cellulosome-like multienzyme complexes. The production of cellulosome-like complexes and the extracellular β-xylosidase activity against 7-xylosyltaxanes might be used to differentiate the genus Cellulosimicrobium from other genera of the family Promicromonosporaceae.

     

Development of a novel engineered antibody targeting <Emphasis Type="Italic">Neisseria</Emphasis> species

Objectives

A Neissaria bacterial pilus sugar, bacillosamine, was synthesized and, for the first time, used as a probe to screen a single-chain variable fragment (scFv).

Results

Four Neisseria, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria sicca and Neisseria subflava, and two negative controls, Streptococcus pneumoniae and Escherichia coli, were tested through ELISA, immunostaining and gold nanoparticle immunological assay. All results indicated that the selected scFv is feasible for the specific detection of Neisseria species via the recognition of bacillosamine.

Conclusions

The recombinant scFv could detect Neisseria strains at 10⁶ CFU/ml.

     

Expression and characterisation of neopullulanase from <Emphasis Type="Italic">Lactobacillus mucosae</Emphasis>

Objectives

To clone and express a neopullulanase gene from Lactobacillus mucosae LM1 in Escherichia coli and characterise the resulting recombinant neopullulanase.

Results

An ORF in L. mucosae corresponding to a neopullulanase was cloned and expressed in E. coli. The predicted amino acid sequence of the neopullulanase contained catalytic sites and conserved motifs that are present in members of the neopullulanase subfamily. The resulting recombinant neopullulanase was efficiently purified by Ni–NTA affinity chromatography. The purified enzyme optimally hydrolyses pullulan at 37 °C and pH 6.0, producing panose as the major reaction product.

Conclusions

To the best of our knowledge, this is the first report of the cloning, expression and characterisation of a neopullulanase gene from a lactic acid bacterium.

     

Codon optimization of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> mating factor alpha prepro-leader to improve recombinant protein production in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objectives

To evaluate different codon optimization parameters on the Saccharomyces cerevisiae-derived mating factor α prepro-leader sequence (MFLS) to improve Candida antarctica lipase B (CAL-B) secretory production in Pichia pastoris.

Results

Codon optimization based on the individual codon usage (ICU) and codon context (CC) design parameters enhanced secretory production of CAL-B to 7 U/ml and 12 U/ml, respectively. Only 3 U/ml was obtained with the wild type sequence while the sequence optimized using both ICU and CC objectives showed intermediate performance of 10 U/ml. These results clearly show that CC is the most relevant parameter for the codon optimization of MFLS in P. pastoris, and there is no synergistic effect achieved by considering both ICU and CC together.

Conclusion

The CC optimized MFLS increased secretory protein production of CAL-B in P. pastoris by fourfold.

     

The bacterial community associated with rose-scented geranium (<Emphasis Type="Italic">Pelargonium graveolens</Emphasis>) leaves responds to anthracnose symptoms

Background

The fungus Colletotrichum is a plant pathogen that causes the anthracnose disease, resulting in huge losses in various crops including the rose-scented geranium (Pelargonium graveolens). Although the bacterial community associated with plants has an important role in the establishment of plant diseases, little is known about what happens in P. graveolens.

Aims

To increase the knowledge about the bacterial community associated with P. graveolens and its relationship with anthracnose disease symptoms.

Methods

Quantitative PCR and high-throughput sequencing were combined to determine the presence of the fungus Colletotrichum and to reveal the bacterial communities associated with different plant parts – root, stem and leaf – and in the rhizosphere and bulk soil, and also to determine the respective bacterial communities associated with P. graveolens leaves symptomatic and asymptomatic for anthracnose disease.

Results

The fungus Colletotrichum was detected in all plant parts and in the surrounding soil. Bacterial communities varied spatially in plants, and the disease symptoms also influenced the composition of the bacterial community. Abundances of operational taxonomic units (OTUs) assigned to the phylum Actinobacteria and to the genus Streptococcus were greatly increased in asymptomatic leaves.

Conclusions

The bacterial community associated to geranium leaves responds to anthracnose symptoms.

     

Plant growth-promoting bacterium <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> FR1 secrets a novel type of extracellular polyhydroxybutyrate polymerase involved in abiotic stress response in plants

Objectives

Identification of novel microbial factors contributing to plant protection against abiotic stress.

Results

The genome of plant growth-promoting bacterium Pseudomonas fluorescens FR1 contains a short mobile element encoding a novel type of extracellular polyhydroxybutyrate (PHB) polymerase (PhbC) associated with a type I secretion system. Genetic analysis using a phbC mutant strain and plants showed that this novel extracellular enzyme is related to the PHB production in planta and suggests that PHB could be a beneficial microbial compound synthesized during plant adaptation to cold stress.

Conclusion

Extracellular PhbC can be used as a new tool for improve crop production under abiotic stress.

     

A versatile <Emphasis Type="Italic">Trichoderma reesei</Emphasis> expression system for the production of heterologous proteins

Objectives

To develop a versatile Trichoderma reesei (teleomorph Hypocrea jecorina) expression system for the high-purity production of heterologous proteins.

Results

The versatile T. reesei expression system is based on xyn1 and xyn2 promoters, A824V transition in XYRI, and a bicomponent carbon source strategy. Red fluorescent protein gene rfp and alkaline endoglucanase EGV gene egv3 from Humicola insolens were used as reporter genes to test our versatile expression system

Conclusions

The versatile T. reesei expression system can be applied to produce heterologous proteins with high purity and high yield.

     

Effect of 1-aminocyclopropane-1-carboxylic acid (ACC)-induced ethylene on <Emphasis Type="Italic">cellulose synthase A</Emphasis> (<Emphasis Type="Italic">CesA</Emphasis>) genes in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L. ‘Nike’) seedlings

Introduction

Cellulose microfibril is a major cell wall polymer that plays an important role in the growth and development of plants. The gene cellulose synthase A (CesA), encoding cellulose synthases, is involved in the synthesis of cellulose microfibrils. However, the regulatory mechanism of CesA gene expression is not well understood, especially during the early developmental stages.

Objective

To identify factor(s) that regulate the expression of CesA genes and ultimately control seedling growth and development.

Methods

The presence of cis-elements in the promoter region of the eight CesA genes identified in flax (Linum usitatissimum L. ‘Nike’) seedlings was verified, and three kinds of ethylene-responsive cis-elements were identified in the promoters. Therefore, the effect of ethylene on the expression of four selected CesA genes classified into Clades 1 and 6 after treatment with 10^?4 and 10^?3 M 1-aminocyclopropane-1-carboxylic acid (ACC) was examined in the hypocotyl of 4–6-day-old flax seedlings.

Results

ACC-induced ethylene either up- or down-regulated the expression of the CesA genes depending on the clade to which these genes belonged, age of seedlings, part of the hypocotyl, and concentration of ACC.

Conclusion

Ethylene might be one of the factors regulating the expression of CesA genes in flax seedlings.

     

Synthesis of rosmarinic acid analogues in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To produce rosmarinic acid analogues in the recombinant Escherichia coli BLRA1, harboring a 4-coumarate: CoA ligase from Arabidopsis thaliana (At4CL) and a rosmarinic acid synthase from Coleus blumei (CbRAS).

Results

Incubation of the recombinant E. coli strain BLRA1 with exogenously supplied phenyllactic acid (PL) and analogues as acceptor substrates, and coumaric acid and analogues as donor substrates led to production of 18 compounds, including 13 unnatural RA analogues.

Conclusion

This work demonstrates the viability of synthesizing a broad range of rosmarinic acid analogues in E. coli, and sheds new light on the substrate specificity of CbRAS.

     

Seroprevalence of <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato and <Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> in Danish horses

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum are able to infect horses. However, the extend to which Danish horses are infected and seroconvert due to these two bacteria is unknown. The aim of the present study was to evaluate the seroprevalence of B. burgdorferi sensu lato and A. phagocytophilum in Danish horses.

Methods

A total of 390 blood samples collected from all major regions of Denmark and with a geographical distribution corresponding to the density of the Danish horse population were analyzed. All samples were examined for the presence of antibodies against B. burgdorferi sensu lato and A. phagocytophilum by the use of the SNAP^®4DX ^® ELISA test.

Results

Overall, 29.0% of the horses were seropositive for B. burgdorferi sensu lato whereas 22.3% were seropositive for A. phagocytophilum.

Conclusions

Antibodies against B burgdorferi sensu lato and A. phagocytophilum are commonly found among Danish horses thus showing that Danish horses are frequently infected by these organisms.

     

<Emphasis Type="Italic">Candida guilliermondii</Emphasis> as a potential biocatalyst for the production of long-chain α,ω-dicarboxylic acids

Objectives

To explore Candida guilliermondii for the production of long-chain dicarboxylic acids (DCA), we performed metabolic pathway engineering aiming to prevent DCA consumption during β-oxidation, but also to increase its production via the ω-oxidation pathway.

Results

We identified the major β- and ω-oxidation pathway genes in C. guilliermondii and performed first steps in the strain improvement. A double pox disruption mutant was created that slowed growth with oleic acid but showed accelerated DCA degradation. Increase in DCA production was achieved by homologous overexpression of a plasmid borne cytochrome P450 monooxygenase gene.

Conclusion

C. guilliermondii is a promising biocatalyst for DCA production but further insight into its fatty acid metabolism is necessary.