HAlign-II: efficient ultra-large multiple sequence alignment and phylogenetic tree reconstruction with distributed and parallel computing

Background

Multiple sequence alignment (MSA) plays a key role in biological sequence analyses, especially in phylogenetic tree construction. Extreme increase in next-generation sequencing results in shortage of efficient ultra-large biological sequence alignment approaches for coping with different sequence types.

Methods

Distributed and parallel computing represents a crucial technique for accelerating ultra-large (e.g. files more than 1 GB) sequence analyses. Based on HAlign and Spark distributed computing system, we implement a highly cost-efficient and time-efficient HAlign-II tool to address ultra-large multiple biological sequence alignment and phylogenetic tree construction.

Results

The experiments in the DNA and protein large scale data sets, which are more than 1GB files, showed that HAlign II could save time and space. It outperformed the current software tools. HAlign-II can efficiently carry out MSA and construct phylogenetic trees with ultra-large numbers of biological sequences. HAlign-II shows extremely high memory efficiency and scales well with increases in computing resource.

Conclusions

THAlign-II provides a user-friendly web server based on our distributed computing infrastructure. HAlign-II with open-source codes and datasets was established at http://lab.malab.cn/soft/halign.

     

Model-based reconstruction of synthetic promoter library in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Objective

To develop an efficient synthetic promoter library for fine-tuned expression of target genes in Corynebacterium glutamicum.

Results

A synthetic promoter library for C. glutamicum was developed based on conserved sequences of the ??10 and ??35 regions. The synthetic promoter library covered a wide range of strengths, ranging from 1 to 193% of the tac promoter. 68 promoters were selected and sequenced for correlation analysis between promoter sequence and strength with a statistical model. A new promoter library was further reconstructed with improved promoter strength and coverage based on the results of correlation analysis. Tandem promoter P70 was finally constructed with increased strength by 121% over the tac promoter. The promoter library developed in this study showed a great potential for applications in metabolic engineering and synthetic biology for the optimization of metabolic networks.

Conclusions

To the best of our knowledge, this is the first reconstruction of synthetic promoter library based on statistical analysis of C. glutamicum.

     

ReToAd: simple method for the rapid replacement of promoters to improve protein production

Objective

To develop a method for fast replacement of promoters to improve protein production.

Results

A method (entitled retreat to advance or “ReToAd”), which includes a deleting PCR and a touchdown PCR, was validated by replacing seven IPTG-inducible promoters with enhanced green fluorescent protein (eGFP). The seven promoters were fully recovered by sequencing only 30 clones. The activity of E. coli harboring ω-transaminase (ω-TA) was increased from 112 U/mg cells (T7 promoter) to 147 U/mg cells (Trc promoter) by combining ReToAd and screening experiments. After screening a library comprising glutamate dehydrogenase (GDH) expressed by different promoters, the activity of E. coli cell harboring Trc-promoter-expressed GDH was ~31-fold higher than that of T7-promoter-expressed GDH.

Conclusions

The “ReToAd” for in situ rapid replacement of promoters was developed and optimized, and one round of “ReToAd” can be completed within 3 days.

     

Determination and optimization of a strong promoter element from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> by using a promoter probe vector

Objective

To construct a promoter probe vector, pBE-bgaB, to screen strong promoters from Bacillus amyloliquefaciens.

Results

266 colonies containing active promoter elements from the genomic DNA of B. amyloliquefaciens were identified. Among these, promoter P41 exhibited the strongest β-Gal activity in Escherichia coli and B. amyloliquefaciens. Sequence analysis showed that promoter P41 contained P _ykuN , a ykuN gene encoding flavodoxin. Optimization of the ribosome-binding site from P41 to P₃₈₂ improved β-Gal activity by ~ 200%.

Conclusion

A new strong promoter for protein expression and genetic engineering of Bacillus species.

     

Native promoters of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> and its application in <Emphasis Type="SmallCaps">l</Emphasis>-lysine production

Objective

To identify useful native promoters of Corynebacterium glutamicum for fine-tuning of gene expression in metabolic engineering.

Results

Sixteen native promoters of C. glutamicum were characterized. These promoters covered a strength range of 31-fold with small increments and exhibited relatively stable activity during the whole growth phase using β-galactosidase as the reporter. The mRNA level and enzymatic activity of the lacZ reporter gene exhibited high correlation (R ² = 0.96) under the control of these promoters. Sequence analysis found that strong promoters had high similarity of the -10 hexamer to the consensus sequence and preference of the AT-rich UP element upstream the -35 region. To test the utility of the promoter library, the characterized native promoters were applied to modulate the sucCD-encoded succinyl-CoA synthetase expression for l-lysine overproduction.

Conclusions

The native promoters with various strengths realize the efficient and precise regulation of gene expression in metabolic engineering of C. glutamicum.

     

Hot spot prediction in protein-protein interactions by an ensemble system

Background

Hot spot residues are functional sites in protein interaction interfaces. The identification of hot spot residues is time-consuming and laborious using experimental methods. In order to address the issue, many computational methods have been developed to predict hot spot residues. Moreover, most prediction methods are based on structural features, sequence characteristics, and/or other protein features.

Results

This paper proposed an ensemble learning method to predict hot spot residues that only uses sequence features and the relative accessible surface area of amino acid sequences. In this work, a novel feature selection technique was developed, an auto-correlation function combined with a sliding window technique was applied to obtain the characteristics of amino acid residues in protein sequence, and an ensemble classifier with SVM and KNN base classifiers was built to achieve the best classification performance.

Conclusion

The experimental results showed that our model yields the highest F1 score of 0.92 and an MCC value of 0.87 on ASEdb dataset. Compared with other machine learning methods, our model achieves a big improvement in hot spot prediction.

Availability

http://deeplearner.ahu.edu.cn/web/HotspotEL.htm.

     

COGcollator: a web server for analysis of distant relationships between homologous protein families

Background

The Clusters of Orthologous Groups (COGs) of proteins systematize evolutionary related proteins into specific groups with similar functions. However, the available databases do not provide means to assess the extent of similarity between the COGs.

Aim

We intended to provide a method for identification and visualization of evolutionary relationships between the COGs, as well as a respective web server.

Results

Here we introduce the COGcollator, a web tool for identification of evolutionarily related COGs and their further analysis. We demonstrate the utility of this tool by identifying the COGs that contain distant homologs of (i) the catalytic subunit of bacterial rotary membrane ATP synthases and (ii) the DNA/RNA helicases of the superfamily 1.

Reviewers

This article was reviewed by Drs. Igor N. Berezovsky, Igor Zhulin and Yuri Wolf.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

The role of Exo70 in vascular smooth muscle cell migration

Background

As a key subunit of the exocyst complex, Exo70 has highly conserved sequence and is widely found in yeast, mammals, and plants. In yeast, Exo70 mediates the process of exocytosis and promotes anchoring and integration of vesicles with the plasma membrane. In mammalian cells, Exo70 is involved in maintaining cell morphology, cell migration, cell connection, mRNA splicing, and other physiological processes, as well as participating in exocytosis. However, Exo70’s function in mammalian cells has yet to be fully recognized. In this paper, the expression of Exo70 and its role in cell migration were studied in a rat vascular smooth muscle cell line A7r5.

Methods

Immunofluorescent analysis the expression of Exo70, α-actin, and tubulin in A7r5 cells showed a co-localization of Exo70 and α-actin, we treated the cells with cytochalasin B to depolymerize α-actin, in order to further confirm the co-localization of Exo70 and α-actin. We analyzed Exo70 co-localization with actin at the edge of migrating cells by wound-healing assay to establish whether Exo70 might play a role in cell migration. Next, we analyzed the migration and invasion ability of A7r5 cells before and after RNAi silencing through the wound healing assay and transwell assay.

Results

The mechanism of interaction between Exo70 and cytoskeleton can be clarified by the immunoprecipitation techniques and wound-healing assay. The results showed that Exo70 and α-actin were co-localized at the leading edge of migrating cells. The ability of A7r5 to undergo cell migration was decreased when Exo70 expression was silenced by RNAi. Reducing Exo70 expression in RNAi treated A7r5 cells significantly lowered the invasion and migration ability of these cells compared to the normal cells. These results indicate that Exo70 participates in the process of A7r5 cell migration.

Conclusions

This research is importance for the study on the pathological process of vascular intimal hyperplasia, since it provides a new research direction for the treatment of cardiovascular diseases such as atherosclerosis and restenosis after balloon angioplasty.

     

A lost opportunity for science: journals promote data sharing in metabolomics but do not enforce it

Introduction

Data sharing is being increasingly required by journals and has been heralded as a solution to the ‘replication crisis’.

Objectives

(i) Review data sharing policies of journals publishing the most metabolomics papers associated with open data and (ii) compare these journals’ policies to those that publish the most metabolomics papers.

Methods

A PubMed search was used to identify metabolomics papers. Metabolomics data repositories were manually searched for linked publications.

Results

Journals that support data sharing are not necessarily those with the most papers associated to open metabolomics data.

Conclusion

Further efforts are required to improve data sharing in metabolomics.

     

Pseudo current density maps of electrophysiological heart,nerve or brain function and their physical basis

Background

In recent years the visualization of biomagnetic measurement data by so-called pseudo current density maps or Hosaka-Cohen (HC) transformations became popular.

Methods

The physical basis of these intuitive maps is clarified by means of analytically solvable problems.

Results

Examples in magnetocardiography, magnetoencephalography and magnetoneurography demonstrate the usefulness of this method.

Conclusion

Hardware realizations of the HC-transformation and some similar transformations are discussed which could advantageously support cross-platform comparability of biomagnetic measurements.