A novel method for single-grain-based metabolic profiling of Arabidopsis seed

Introduction

In plant metabolomics, metabolite contents are often normalized by sample weight. However, accurate weighing of very small samples, such as individual Arabidopsis thaliana seeds (approximately 20 µg), is difficult, which may lead to irreproducible results.

Objectives

We aimed to establish alternative normalization methods for seed-grain-based comparative metabolomics of A. thaliana.

Methods

Arabidopsis thaliana seeds were assumed to have a prolate spheroid shape. Using a microscope image of each seed, the lengths of major and minor axes were measured by fitting a projected 2-dimensional shape of each seed as an ellipse. Metabolic profiles of individual diploid or tetraploid A. thaliana seeds were measured by our highly sensitive protocol (“widely targeted metabolomics”) that uses liquid chromatography coupled with tandem quadrupole mass spectrometry. Mass spectrometric analysis of 1 µL of solution extract identified more than 100 metabolites. The data were normalized by various seed-size measures, including seed volume (single-grain-based analysis). For comparison, metabolites were extracted from 4 mg of diploid and tetraploid A. thaliana seeds and their metabolic profiles were analyzed by normalization of weight (weight-based analysis).

Results

A small number of metabolites showed statistically significant differences in the single-grain-based analysis compared to weight-based analysis. A total of 17 metabolites showed statistically different accumulation between ploidy types with similar fold changes in both analyses.

Conclusion

Seed-size measures obtained by microscopic imaging were useful for data normalization. Single-grain-based analysis enables evaluation of metabolism of each seed and elucidates the metabolic profiles of precious bioresources by using small amounts of samples.

     

Evaluation of lytic bacteriophages for control of multidrug-resistant <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium

Background

The emergence of antibiotic-resistant bacteria can cause serious clinical and public health problems. This study describes the possibility of using bacteriophages as an alternative agent to control multidrug-resistant Salmonella Typhimurium.

Methods

The potential lytic bacteriophages (P22-B1, P22, PBST10, PBST13, PBST32, and PBST 35) were characterized by morphological property, heat and pH stability, optimum multiplicity of infection (MOI), and lytic activity against S. Typhimurium KCCM 40253, S. Typhimurium ATCC 19585, ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and S. Typhimurium CCARM 8009.

Results

P22-B1 and P22 belong to Podoviridae family and PBST10, PBST13, PBST32, and PBST 35 show a typical structure with polyhedral head and long tail, belonging to Siphoviridae family. Salmonella bacteriophages were highly stable at the temperatures (< 60 °C) and pHs (5.0–11.0). The reduction rates of host cells were increased at the MOI-dependent manner, showing the highest reduction rate at MOI of 10. The host cells were most effectively reduced by P22, while P22-B1 showed the least lytic activity. The ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585, and clinically isolated antibiotic-resistant S. Typhimurium CCARM 8009 were resistant to ciprofloxacin, levofloxacin, norfloxacin, and tetracycline. P22 showed the highest lytic activity against S. Typhimurium KCCM 40253 (> 5 log reduction), followed by S. Typhimurium ATCC 19585 (4 log reduction) and ciprofloxacin-induced antibiotic-resistant S. Typhimurium ATCC 19585 (4 log reduction).

Conclusion

The results would provide vital insights into the application of lytic bacteriophages as an alternative therapeutics for the control of multidrug-resistant pathogens.

     

Metabolic response of porcine colon explants to in vitro infection by <Emphasis Type="Italic">Brachyspira hyodysenteriae</Emphasis>: a leap into disease pathophysiology

Introduction

Swine dysentery caused by Brachyspira hyodysenteriae is a production limiting disease in pig farming. Currently antimicrobial therapy is the only treatment and control method available.

Objective

The aim of this study was to characterize the metabolic response of porcine colon explants to infection by B. hyodysenteriae.

Methods

Porcine colon explants exposed to B. hyodysenteriae were analyzed for histopathological, metabolic and pro-inflammatory gene expression changes.

Results

Significant epithelial necrosis, increased levels of l-citrulline and IL-1α were observed on explants infected with B. hyodysenteriae.

Conclusions

The spirochete induces necrosis in vitro likely through an inflammatory process mediated by IL-1α and NO.

     

Dynamic changes of histone H3 lysine 9 following trimethylation in bovine oocytes and pre-implantation embryos

Objectives

We have examined dynamic changes of histone H3 lysine 9 following trimethylation (H3K9me3), the mRNA expression levels of SUV39H1 and SUV39H2 in bovine oocytes and the role in the development of in vitro fertilization (IVF) pre-implantation embryos.

Results

There were strong H3K9me3 signals in germinal vesicle (GV) oocytes but no signals in MII oocytes. H3K9me3 signals were maintained during IVF pre-implantation embryo development. SUV39H1 and SUV39H2 showed significantly higher mRNA expression levels in GV oocytes than MII oocytes (P < 0.01). SUV39H1 showed high mRNA expression level in two-cell embryos, however, SUV39H2 showed high mRNA expression level in four-cell embryos. In other development stage, SUV39H1 and SUV39H2 showed low expression levels.

Conclusion

Bovine IVF pre-implantation embryos maintain strong H3K9me3 signals and SUV39H1 and SUV39H2 are highly expressed at the early development stage of pre-implantation embryos.

     

The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Objective

α-Galactosidases are widely used in many fields. It is necessary to improve the production of enzymes through microbiological processes. The aim of this study was to construct recombinant Aspergillus niger strains with high α-galactosidase production.

Results

Two recombinant A. niger strains were constructed: AB and AGB. The recombinant AB strain contained the α-galactosidase aglB gene from A. niger with its native AglB signal peptide regulated by the glucoamylase promoter. In the AGB recombinant strain, the AglB signal peptide was replaced with the glucoamylase (GlaA) signal peptide. The extracellular maximum α-galactosidase activity of the AGB strain was 215.7 U/ml and that of the AB strain was 9.8 U/mL. The optimal conditions for α-galactosidase were pH 3.5 and 35 °C.

Conclusions

The GlaA signal peptide substantially increased the yield of secreted α-galactosidase in A. niger. This recombinant strain holds great potential for industrial applications.

     

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.

     

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.

     

A constitutive expression system for <Emphasis Type="Italic">Pichia pastoris</Emphasis> based on the <Emphasis Type="Italic">PGK1</Emphasis> promoter

Objectives

To develop a new vector for constitutive expression in Pichia pastoris based on the endogenous glycolytic PGK1 promoter.

Results

P. pastoris plasmids bearing at least 415 bp of PGK1 promoter sequences can be used to drive plasmid integration by addition at this locus without affecting cell growth. Based on this result, a new P. pastoris integrative vector, pPICK2, was constructed bearing some features that facilitate protein production in this yeast: a ~620 bp PGK1 promoter fragment with three options of restriction sites for plasmid linearization prior to yeast transformation: a codon-optimized α-factor secretion signal, a new polylinker, and the kan marker for vector propagation in bacteria and selection of yeast transformants.

Conclusions

A new constitutive vector for P. pastoris represents an alternative platform for recombinant protein production and metabolic engineering purposes.

     

Construction and co-expression of polycistronic plasmids encoding bio-degumming-related enzymes to improve the degumming process of ramie fibres

Objectives

To research the inherent properties of the co-expression of three types degumming-related enzymes and breed more powerful degumming strains.

Results

Six tandem multimers of the pectate lyase gene, the xylanase gene, and the endo-1,4-β-mannanase gene, which are essential for degumming process, were co-expressed and evaluated in Escherichia coli BL21(DE3). The xyl91 gene had a synergistic effect with endo-1,4-β-mannanase and pectate lyase from DCE-01, when xyl gene was replaced with xyl91 in the multimer. The recombinant pET-pxm(91x) was selected and transformed into the original degumming strain DCE-01, which led to an enzymatic activity improvement. Furthermore, the weight loss, reducing sugar and COD value of the sample treated with the new engineered strain pET-pxm(91x)/DCE-01 increased to 22.5 %, 460 mg ml^?1 and 4.9, respectively.

Conclusions

The co-expression of degumming-related enzyme genes may be applied in industrial tests and represents a novel direction for bio-degumming research.

     

Inhibitory effects of YCW and MOS from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Salmonella pullorum</Emphasis> adhesion to Caco-2 cells

Background

For many years, yeast cell walls (YCW) and mannan oligosaccharides (MOS) have been used as alternatives to antibiotics and health feed additives to enhance the growth performance and health of food animals. In the present study, the inhibitory effects of YCWand MOS on the adhesion of enteropathogenic bacteria to intestinal epithelial cells were tested.

Methods

YCW and MOS were extracted from Saccharomyces cerevisiae (XM 0315), and the morphology of YCW and MOS bound to pathogenic bacteria was observed by scanning electron microscopy (SEM). Real-time fluorescent quantitative PCR was used to quantitatively analyze the effects of YCW and MOS on the adhesion of Escherichia coli (CVCC3367) and Salmonella pullorum (CVCC520) to Caco-2 cells.

Results

The results showed that YCW inhibited E. coli and S. pullorum binding to Caco-2 cells by 95% and 74%, respectively, whereas MOS prevented E. coli and S. pullorum binding by 67% and 50%, respectively.

Conclusions

These data suggest that YCW has a stronger ability than MOS to inhibit pathogenic bacteria from adhering to Caco-2 cells in vitro.

     

Evaluation of the Clearview<Superscript>®</Superscript> malaria pLDH malaria rapid diagnostic test in a non-endemic setting

Background

Malaria Rapid Diagnostic Tests (RDTs) are widely used to diagnose malaria. The present study evaluated a new RDT, the Clearview^® Malaria pLDH test targeting the pan-Plasmodium antigen lactate dehydrogenase (pLDH).

Methods

The Clearview^® Malaria pLDH test was evaluated on fresh samples obtained in returned international travellers using microscopy corrected by PCR as the reference method. Included samples were Plasmodium falciparum (139), Plasmodium vivax (22), Plasmodium ovale (20), Plasmodium malariae (7), and 102 negative.

Results

Overall sensitivity for the detection of Plasmodium spp was 93.2%. For P. falciparum, the sensitivity was 98.6%; for P. vivax, P. ovale and P. malariae, overall sensitivities were 90.9%, 60.0% and 85.7% respectively. For P. falciparum and for P. vivax, the sensitivities increased to 100% at parasite densities above 100/μl. The specificity was 100%. The test was easily to perform and the result was stable for at least 1 hour.

Conclusion

The Clearview^® Malaria pLDH was efficient for the diagnosis of malaria. The test was very sensitive for P. falciparum and P. vivax detection. The sensitivities for P. ovale and P. malariae were better than other RDTs

     

Secreted expression of truncated capsid protein from porcine circovirus type 2 in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To achieve secreted expression of the truncated capsid protein from porcine circovirus type 2 (PCV2) in Pichia pastoris.

Results

A truncated cap gene (tcap) with a deleted N-terminal nuclear localization signal was optimized and synthesized. Effective secreted expression was achieved in P. pastoris GS115. The high-productive recombinant strain for tCap was grown in a 5 l bioreactor and the productivity of tCap in supernatant reached 250 μg/ml. Furthermore, serum antibody test demonstrated that adjuvant-assisting tCap induced a significant increase of specific PCV2-Cap antibody over time in mice and a similar antibody level in pigs compared with a commercial Cap-based subunit vaccine.

Conclusion

This work establishes a secreted expression strategy in P. pastoris for the production of PCV2 Cap with superior bioactivity, and this strategy might provide potential uses in developing Cap-based subunit vaccine in the future.

     

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.

     

Fine mapping a self-fertility locus in perennial ryegrass

Key message

A self-fertility locus was fine mapped to a 1.6 cM region on linkage group 5 in a perennial ryegrass population. This locus was the main determinant of pollen self-compatibility.

Abstract

In grasses, self-incompatibility (SI) is characterized by a two-loci gametophytic (S and Z) mechanism acting together in the recognition and inhibition of self-pollen. Mutations affecting the expression of SI have been reported in a few grass species. In perennial ryegrass (Lolium perenne L.), a mutation independent from S and Z, and mapping on linkage group 5 (LG 5), was previously reported to produce self-fertile plants. Here, we describe fine mapping of the self-fertility (SF) gene in a perennial ryegrass population and determine whether there is any effect of other genomic regions on the pollen compatibility. The phenotypic segregation of SF showed a bimodal distribution with one mean at 49% pollen compatibility and the other at 91%. Marker-trait association analysis showed that only markers on LG 5 were significantly associated with the trait. A single gene model explained 82% of the observed variability and no effects of the other regions were detected. Using segregation and linkage analysis, the SF locus was located to a 1.6 cM region on LG 5. The flanking marker sequences were aligned to rice and Brachypodium distachyon reference genomes to estimate the physical distance. We provide markers tightly linked to SF that can be used for introgression of this trait into advanced breeding germplasm. Moreover, our results represent a further step towards the identification of the SF gene in LG 5.

     

Asymmetric reduction of ketopantolactone using a strictly (<Emphasis Type="Italic">R</Emphasis>)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy

Objectives

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].

Results

The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.

Conclusions

Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

     

Correction to: Neighbourhood stories: role of neighbour identity,spatial location and order of arrival in legume and non-legume initial interactions

Background and aims

We characterized fungal endophytes of seeds of invasive, non-native Phragmites from three sites in the Great Lakes region to determine if fungal symbiosis could contribute to invasiveness through their effects on seed germination and seedling growth.

Methods

Field-collected seeds were surface sterilized and plated on agar to culture endophytes for ITS sequencing. Prevalence of specific endophytes from germinated and non-germinated seeds, and from seedlings, was compared.

Results

One-third of 740 seeds yielded endophyte isolates. Fifteen taxa were identified with Alternaria sp. representing 54% of all isolates followed by Phoma sp. (21%) and Penicillium corylophilum (12%). Overall germination of seeds producing an isolate (36%) was significantly higher than seeds not producing an isolate (20%). Penicillium in particular was strongly associated with increased germination of seeds from one site. Sixty-three isolates and 11 taxa were also obtained from 30 seedlings where Phoma, Penicillium and Alternaria respectively were most prevalent. There was a significant effect of isolating an endophyte from the seed on seedling growth.

Conclusions

These results suggest that many endophyte taxa are transmitted in seeds and can increase seed germination and seedling growth of invasive Phragmites. The role of fungal endophytes in host establishment, growth and invasiveness in nature requires further research.

     

Molecular cloning and characterization of <Emphasis Type="Italic">α</Emphasis>-amylase/subtilisin inhibitor from rhizome of <Emphasis Type="Italic">Ligusticum chuanxiong</Emphasis>

Objectives

To clone and characterize a novel bi-functional α-amylase/subtilisin inhibitor (LASI) from the rhizome of Ligusticum chuanxiong, a traditional Chinese medicine.

Results

The LASI showed strong homology with members of the Kunitz trypsin inhibitor family. Its putative amino acid sequence has a 40 % identity with that of the α-amylase/subtilisin inhibitor from rice. LASI gene without signal peptide was expressed in E. coli Rosetta. After purification, the recombinant LASI protein was inhibitory against not only α-amylase from porcine pancreas, Helicoverpa armigera, Spodoptera litura and Plutella xylostella, but also subtilisin A, but not against trypsin or chymotrypsin. In addition, the expression level of LASI in rhizome was higher than that in leaf and LASI expression was enhanced by salt, chilling and drought treatment.

Conclusions

This is the first member of the Kunitz-protease inhibitor family identified in traditional Chinese medicine and it might be involved in the plant defense responses against lepidopterous pests, microorganisms and abiotic stresses.