Seasonal variability of some biochemical parameters in the whitefish (<Emphasis Type="Italic">Coregonus muksun</Emphasis> and <Emphasis Type="Italic">Coregonus lavaretus</Emphasis>)

The base levels and seasonal variability of ethoxyresorufin-O-deethylase (EROD) activity, glutathione-S-transferase (GST) activity, reduced glutathione concentration (GSH) and phospholipids concentration (PhL) of Coregonus lavaretus (Linnaeus, 1758) and Coregonus muksun (Pallas, 1814) were investigated. The relationships between this biochemical markers, hepatosomatic index and seasonal growth were also examined.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Bile salt hydrolase-mediated inhibitory effect of <Emphasis Type="Italic">Bacteroides ovatus</Emphasis> on growth of <Emphasis Type="Italic">Clostridium difficile</Emphasis>

Clostridium difficile infection (CDI) is one of the most common nosocomial infections. Dysbiosis of the gut microbiota due to consumption of antibiotics is a major contributor to CDI. Recently, fecal microbiota transplantation (FMT) has been applied to treat CDI. However, FMT has important limitations including uncontrolled exposure to pathogens and standardization issues. Therefore, it is necessary to evaluate alternative treatment methods, such as bacteriotherapy, as well as the mechanism through which beneficial bacteria inhibit the growth of C. difficile. Here, we report bile acid-mediated inhibition of C. difficile by Bacteroides strains which can produce bile salt hydrolase (BSH). Bacteroides strains are not commonly used to treat CDI; however, as they comprise a large proportion of the intestinal microbiota, they can contribute to bile acid-mediated inhibition of C. difficile. The inhibitory effect on C. difficile growth increased with increasing bile acid concentration in the presence of Bacteroides ovatus SNUG 40239. Furthermore, this inhibitory effect on C. difficile growth was significantly attenuated when bile acid availability was reduced by cholestyramine, a bile acid sequestrant. The findings of this study are important due to the discovery of a new bacterial strain that in the presence of available bile acids inhibits growth of C. difficile. These results will facilitate development of novel bacteriotherapy strategies to control CDI.     

Increasing seed oil content and altering oil quality of <Emphasis Type="Italic">Brassica napus</Emphasis> L. by over-expression of diacylglycerol acyltransferase 1 (<Emphasis Type="Italic">SsDGAT1</Emphasis>) from <Emphasis Type="Italic">Sapium sebiferum</Emphasis> (L.) Roxb.

Sapium sebiferum (L.) Roxb. [S. sebiferum] is one of the most important woody oil trees and traditional herbal medicines in China. Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA acting as a key enzyme at the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Although at least 61 DGAT1 sequences from 48 organisms have been identified, until now there have been almost no reports on the DGAT1 gene in S. sebiferum (SsDGAT1). In an attempt to clarify SsDGAT1’s function, we cloned the CDS (coding sequence) of SsDGAT1 by RACE (rapid amplification of cDNA ends) technology. The full-length CDS of SsDGAT1 contains 1524 bp, encoding a protein of 507 amino acids. Even recombinant SsDGAT1 was able to restore TAG biosynthesis in the yeast strain S. cerevisiae H1246 TAG-deficient mutant. Moreover, when SsDGAT1 was placed under the control of 35S promoter in Brassica napus L. W10, the total oil content of transgenic rape plants showed an increase of 12.3–14.7 % compared with the wild type. In addition, the transgenic rapeseed with heterologous expression of SsDGAT1 showed an alteration in seed oil composition overall: a significant decrease in oleic acid levels but a tendency towards an increase in linolenic acid levels. The result confirmed that SsDGAT1 may be involved in flux control of oil biosynthesis and could be used specifically to manipulate and improve oil content and composition in plants. These experimental findings suggest that we might be able to develop a plant high in industrial oils by over-expression of SsDGAT1 in S. sebiferum.     

Functional role of <Emphasis Type="Italic">oppA</Emphasis> encoding an oligopeptide-binding protein from <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> Ren in bile tolerance

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and some L. salivarius strains are considered as probiotics. Bile tolerance is a crucial property for probiotic bacteria to survive the transit through the GIT and exert their beneficial effects. In this work, the functional role of oppA encoding an oligopeptide transporter substrate-binding protein from L. salivarius Ren in bile salt tolerance was investigated. In silico analysis revealed that the oppA gene encodes a 61.7-kDa cell surface-anchored hydrophilic protein with a canonical lipoprotein signal peptide. Homologous overexpression of OppA was shown to confer 20-fold higher tolerance to 0.5 % oxgall in L. salivarius Ren. Furthermore, the recombinant strain exhibited 1.8-fold and 3.6-fold higher survival when exposed to the sublethal concentration of sodium taurocholate and sodium taurodeoxycholate, respectively, while no significant change was observed when exposed to sodium glycocholate and sodium glycodeoxycholate (GDCA). Our results indicate that OppA confers specific resistance to taurine-conjugated bile salts in L. salivarius Ren. In addition, the OppA overexpression strain also showed significant increased resistance to heat and salt stresses, suggesting the protective role of OppA against multiple stresses in L. salivarius Ren.     

The <Emphasis Type="Italic">Adh</Emphasis> locus and adaptation of <Emphasis Type="Italic">cn</Emphasis> and <Emphasis Type="Italic">vg</Emphasis> mutants in experimental populations of <Emphasis Type="Italic">Drosophila melanogaster MEIG</Emphasis>

A complex study on the adaptation of cn and vn mutants and the allozymes of alcoholdehydrogenase (ADH) was carried out in initially pure lines, and their panmixia populations during exchange of the mutant genotype with that of wild-type flies (C-S) and D) through saturating crossings. The relative adaptation of the genotypes was estimated by their effect on reproductive efficiency in the experimentally obtained population. Fecundity, lifespan, and the resistance of the studied genotypes to hyperthermia were investigated individually. It was shown that the high level of adaptation of the cn mutants and the low level of adaptation of the vg mutants was correlated with the presence of different ADH allozymes. In the studied population, the F-allozyme of ADH accompanied the vg mutation, while the S-allozyme of the enzyme was detected in cn mutants. Saturating crossings of C-S(Adh ^S)×vg(Adh ^F) and D(Adh ^F) × cn(Adh ^S), along with the parallel determination of the allele composition of the Adh locus, demonstrated that the complete substitution of the F-allozyme of ADH in the vg mutants by the S-allozyme in D flies, as well as the substitution of the S-allozyme of ADH in the cn mutants by the F-allozyme in D flies was realized only after the 15th–20th backcrosses. These results favor the coadaptation of cn and vg marker genes with alleles of the Adh locus and indicate the important role of the latter in the adaptation of genotypes. In the studied population, selection acted primarily against the vg mutants, which were inferior to the cn mutants, and heterozygote genotypes in indices of the main adaptation components.     

<Emphasis Type="Italic">Lactobacillus</Emphasis> spp. impair the ability of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> FBUNT to adhere to and invade Caco-2 cells

Objectives

The objective of this study was to evaluate the ability of Lactobacillus curvatus CRL705, CRL1532, and CRL1533 and Lactobacillus sakei CRL1613 to survive under simulated gastrointestinal conditions. Moreover, a microencapsulation approach was proposed to improve gastrointestinal survival. Finally, experiments were performed to demonstrate that Lactobacillus spp. can modulate the ability of Listeria monocytogenes FBUNT to adhere to and invade Caco-2 cells.

Results

Lactobacillus strains were encapsulated in alginate beads to enhance the survival of bacteria under in vitro gastrointestinal conditions. All strains hydrolyzed bile salts using chenodeoxycholic acid as a substrate and adhered to Caco-2 cells. Cell-free supernatants (CFSs) showed antimicrobial activity against L. monocytogenes as demonstrated by agar diffusion assays. The average percentages of L. monocytogenes adhesion decreased from 67.74 to 41.75 and 38.7% in the presence of 50 and 90% (v/v), respectively, for all CFSs tested. The highest concentrations of CFSs completely inhibited the L. monocytogenes invasion of Caco-2 cells.

Conclusions

The studied Lactobacillus strains have protective effects against the adhesion and invasion of L. monocytogenes FBUNT. Alginate encapsulation of these bacteria improved gastrointestinal tolerance such that they could be further studied as potential probiotics against intestinal pathogenic bacteria.

     

Acaricidal active fractions from acetone extract of <Emphasis Type="Italic">Aloe vera</Emphasis> L. against <Emphasis Type="Italic">Tetranychus cinnabarinus</Emphasis> and <Emphasis Type="Italic">Panonychus citri</Emphasis>

This study aimed to isolate acaricidal active fractions from acetone extract of Aloe vera L. and investigate the toxicity of these fractions against Tetranychus cinnabarinus (T. cinnabarinus) and Panonychus citri (P. citri). Acetone extract of A. vera L. was isolated by immersing in acetone for 72 h, and diverse fractions were fractionated by column chromatography. The acaricidal activity of each fractions was evaluated by corrected mortality of T. cinnabarinus through slide-dip bioassay. The 8th and 13th fractions of acetone extract with good acaricidal activity were indentified by LC/MS, and the toxicity of these two fractions to T. cinnabarinus and P. citri was identified by regression analysis. Acetone extract of A. vera L. exhibited obvious acaricidal activity, from which a total of 18 fractions were isolated. The 8th and 13th fractions with strong acaricidal activity against T. cinnabarinus were identified to be 3-O-alpha-d-mannopyranosyl-d-mannopyranose (OAMM) and aloe emodin. When compared with spirodiclofen, both OAMM and aloe emodin exhibited higher toxicity to T. cinnabarinus, while only OAMM exhibited a higher toxicity to P. citri (P < 0.05). OAMM and aloe emodin isolated from acetone extract of A. vera L. exhibited obvious acaricidal activities against T. cinnabarinus and P. citri.     

Phylogeography of the Macaronesian Lettuce Species <Emphasis Type="Italic">Lactuca watsoniana</Emphasis> and <Emphasis Type="Italic">L. palmensis</Emphasis> (Asteraceae)

The phylogenetic relationships and phylogeography of two relatively rare Macaronesian Lactuca species, Lactuca watsoniana (Azores) and L. palmensis (Canary Islands), were, until this date, unclear. Karyological information of the Azorean species was also unknown. For this study, a chromosome count was performed and L. watsoniana showed 2n = 34. A phylogenetic approach was used to clarify the relationships of the Azorean endemic L. watsoniana and the La Palma endemic L. palmensis within the subtribe Lactucinae. Maximum parsimony, Maximum likelihood and Bayesian analysis of a combined molecular dataset (ITS and four chloroplast DNA regions) and molecular clock analyses were performed with the Macaronesian Lactuca species, as well as a TCS haplotype network. The analyses revealed that L. watsoniana and L. palmensis belong to different subclades of the Lactuca clade. Lactuca watsoniana showed a strongly supported phylogenetic relationship with North American species, while L. palmensis was closely related to L. tenerrima and L. inermis, from Europe and Africa. Lactuca watsoniana showed four single-island haplotypes. A divergence time estimation of the Macaronesian lineages was used to examine island colonization pathways. Results obtained with BEAST suggest a divergence of L. palmensis and L. watsoniana clades c. 11 million years ago, L. watsoniana diverged from its North American sister species c. 3.8 million years ago and L. palmensis diverged from its sister L. tenerrima, c. 1.3 million years ago, probably originating from an African ancestral lineage which colonized the Canary Islands. Divergence analyses with *BEAST indicate a more recent divergence of the L. watsoniana crown, c. 0.9 million years ago. In the Azores colonization, in a stepping stone, east-to-west dispersal pattern, associated with geological events might explain the current distribution range of L. watsoniana.     

Overexpression of <Emphasis Type="Italic">SsDGAT2</Emphasis> from <Emphasis Type="Italic">Sapium sebiferum</Emphasis> (L.) Roxb Increases Seed Oleic Acid Level in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Sapium sebiferum (L.) Roxb is one of the most important oil trees in China. Diacylglycerol acyltransferases (DGATs) esterify sn-1, 2-diacylglycerol with a long-chain fatty acyl-CoA, the last step and the rate-limiting step of triacylglycerol (TAG) biosynthesis in prokaryotic and eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the SsDGAT2 gene had not been reported to date. To clarify the function of SsDGAT2, we cloned the CDS (rapid amplification of cDNA end) of SsDGAT2 by RACE technology. The full-length CDS of SsDGAT2 contains 1011 bp and encodes a protein of 336 amino acids. Recombinant SsDGAT2 restored TAG biosynthesis to the yeast strain Saccharomyces cerevisiae H1246 TAG-deficient mutant and preferentially incorporated unsaturated C18 fatty acids into lipids. To investigate the biotechnological potential of SsDGAT2, it was expressed under the control of the 35S promoter in Arabidopsis Col-4. The oleic acid content increased by 50 % in transgenic plants relative to the control. The results indicated that most of the oleic acid increase was at the expense of linolenic acid (18:3) content, which suggests that high-oleic-acid-content seeds can be created by the overexpression of SsDGAT2 in S. sebiferum (L.) Roxb.     

Characteristic of Polysaccharide Complexes from <Emphasis Type="Italic">Centaurea scabiosa</Emphasis> L. and <Emphasis Type="Italic">Centaurea pseudomaculosa</Emphasis> Dobrocz.

We characterized polysaccharide complexes from Centaurea scabiosa L. and Centaurea pseudomaculosа Dobrocz. We proposed the technique of sequential selection of water-soluble polysaccharides and pectin substances from the aerial parts of studied objects. We have discovered that the content of water-soluble polysaccharides in the aerial parts of C. scabiosa was 2.8 times higher (2.7 ± 0.3%, n = 3) than in C. pseudomaculosа (0.97 ± 0.50%, n = 3). The content of pectin substances in the aerial parts of C. scabiosa was 2 times higher (7.6 ± 0.4%, n = 3) than in C. pseudomaculosа (3.9 ± 0.3%, n = 3). The residues of D-galacturonic acid, L-rhamnose, D-xylose, D-mannose, D-glucose, and D-galactose are the monomeric units of polysaccharide complexes from C. scabiosa and C. pseudomaculosa. Using ion-exchange chromatography, three polysaccharide fractions (molecular weights 667, 722, and 1027 kDa), whose monomer units are D-galacturonic acid, L-rhamnose, D-galactose, D-xylose, and D-glucose were isolated from the water-soluble polysaccharides of C. scabiosa.     

In vitro probiotic potential of <Emphasis Type="Italic">Lactobacillus delbreuckii</Emphasis> subsp. <Emphasis Type="Italic">bulgaricus</Emphasis> F18 isolated from homemade butter

Present study was carried out to evaluate a new bacterial strain, Lactobacillus delbreuckii subsp. bulgaricus F18 as probiotic strain. L. delbreuckii subsp. bulgaricus F18 was isolated from homemade butter and identified by conventional and molecular techniques. The 16S rRNA sequence of the isolate was registered in National Centre for Biotechnology Information (NCBI) under accession number KT865224. In the present study, L. delbreuckii subsp. bulgaricus F18 exhibited highest viable counts against acid tolerance or low pH tolerance (at pH 1 after 2h of incubation), bile tolerance (conc. 1%), autoaggregation (68%), cell surface hydrophobicity against O-xylene (33.9%), antimicrobial activity against various food borne pathogens (inhibition = 100%), antibiotic sensitivity following standard test methods suggested by various research workers.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.