A Review of the Functionality of Probiotics in the Larviculture Food Chain

During the past two decades, the use of probiotics as an alternative to the use of antibiotics has shown to be promising in aquaculture, particularly in fish and shellfish larviculture. This article reviews the studies on probiotics in larviculture, focusing on the current knowledge of their in vivo mechanisms of action. The article highlights that the in vivo mechanisms of action largely remain to be unravelled. Several methodologies are suggested for further in vivo research, including studies on gut microbiota composition, the use of gnotobiotic animals as test models, and the application of molecular techniques to study host–microbe and microbe–microbe interactions.     

Ligation of Signal Inhibitory Receptor on Leukocytes-1 Suppresses the Release of Neutrophil Extracellular Traps in Systemic Lupus Erythematosus

Neutrophil extracellular traps (NETs) have been implicated in the pathogenesis of systemic Lupus erythematosus (SLE), since netting neutrophils release potentially immunogenic autoantigens including histones, LL37, human neutrophil peptide (HNP), and self-DNA. In turn, these NETs activate plasmacytoid dendritic cells resulting in aggravation of inflammation and disease. How suppression of NET formation can be targeted for treatment has not been reported yet. Signal Inhibitory Receptor on Leukocytes-1 (SIRL-1) is a surface molecule exclusively expressed on phagocytes. We recently identified SIRL-1 as a negative regulator of human neutrophil function. Here, we determine whether ligation of SIRL-1 prevents the pathogenic release of NETs in SLE. Peripheral blood neutrophils from SLE patients with mild to moderate disease activity and healthy donors were freshly isolated. NET release was assessed spontaneously or after exposure to anti-neutrophil antibodies or plasma obtained from SLE patients. The formation of NETs was determined by microscopic evaluation using DNA dyes and immunostaining of NET components, as well as by live cell imaging. We show that SLE neutrophils spontaneously release NETs. NET formation is enhanced by stimulation with antibodies against LL37. Inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and MEK-ERK signaling prevents NET release in response to these antibodies. Signaling via the inhibitory receptor SIRL-1 was induced by ligation with anti-SIRL-1 specific antibodies. Both spontaneous and anti-neutrophil antibody-induced NET formation is suppressed by engagement of SIRL-1. Furthermore, NET release by healthy neutrophils exposed to SLE plasma is inhibited by SIRL-1 ligation. Thus, SIRL-1 engagement can dampen spontaneous and anti-neutrophil antibody-induced NET formation in SLE, likely by suppressing NAPDH oxidase and MEK-ERK activity. Together, these findings reveal a regulatory role for SIRL-1 in NET formation, potentially providing a novel therapeutic target to break the pathogenic loop in SLE.     

Tuning the Fano Resonance Between Localized and Propagating Surface Plasmon Resonances for Refractive Index Sensing Applications

Localized and propagating surface plasmon resonances are known to show very pronounced interactions if they are simultaneously excited in the same nanostructure. Here, we study the Fano interference that occurs between localized surface plasmon resonance (LSPR) and propagating surface plasmon polariton (SPP) modes by means of phase-sensitive spectroscopic ellipsometry. The sample structures consist of periodic gratings of gold nanodisks on top of a continuous gold layer and a thin dielectric spacer, in which the structural dimensions were tuned in such a way that the dipolar LSPR mode and the propagating SPP modes are excited in the same spectral region. We observe pronounced anti-crossing and strongly asymmetric line shapes when both modes move to each other’s vicinity, accompanied of largely increased phase differences between the respective plasmon resonances. Moreover, we show that the anti-crossing can be exploited to increase the refractive index sensitivity of the localized modes dramatically, which result in largely increased values for the figure-of-merit which reaches values between 24 and 58 for the respective plasmon modes.     

Role of Porins in Sensitivity of Escherichia coli to Antibacterial Activity of the Lactoperoxidase Enzyme System

Lactoperoxidase is an enzyme that contributes to the antimicrobial defense in secretory fluids and that has attracted interest as a potential biopreservative for foods and other perishable products. Its antimicrobial activity is based on the formation of hypothiocyanate (OSCN⁻) from thiocyanate (SCN⁻), using H₂O₂ as an oxidant. To gain insight into the antibacterial mode of action of the lactoperoxidase enzyme system, we generated random transposon insertion mutations in Escherichia coli MG1655 and screened the resultant mutants for an altered tolerance of bacteriostatic concentrations of this enzyme system. Out of the ca. 5,000 mutants screened, 4 showed significantly increased tolerance, and 2 of these had an insertion, one in the waaQ gene and one in the waaO gene, whose products are involved in the synthesis of the core oligosaccharide moiety of lipopolysaccharides. Besides producing truncated lipopolysaccharides and displaying hypersensitivity to novobiocin and sodium dodecyl sulfate (SDS), these mutants were also shown by urea-SDS-polyacrylamide gel electrophoresis analysis to have reduced amounts of porins in their outer membranes. Moreover, they showed a reduced degradation of p-nitrophenyl phosphate and an increased resistance to ampicillin, two indications of a decrease in outer membrane permeability for small hydrophilic solutes. Additionally, ompC and ompF knockout mutants displayed levels of tolerance to the lactoperoxidase system similar to those displayed by the waa mutants. These results suggest that mutations which reduce the porin-mediated outer membrane permeability for small hydrophilic molecules lead to increased tolerance to the lactoperoxidase enzyme system because of a reduced uptake of OSCN⁻.     

Using small molecule complexes to elucidate features of photosynthetic water oxidation

The molecular oxygen produced in photosynthesis is generated via water oxidation at a manganese-calcium cluster called the oxygen-evolving complex (OEC). While studies in biophysics, biochemistry, and structural and molecular biology are well known to provide deeper insight into the structure and workings of this system, it is often less appreciated that biomimetic modelling provides the foundation for interpreting photosynthetic reactions. The synthesis and characterization of small model complexes, which either mimic structural features of the OEC or are capable of providing insight into the mechanism of O2 evolution, have become a vital contributor to this scientific field. Our group has contributed to these findings in recent years through synthesis of model complexes, spectroscopic characterization of these systems and probing the reactivity in the context of water oxidation. In this article we describe how models have made significant contributions ranging from understanding the structure of the water-oxidation centre (e.g. contributions to defining a tetrameric Mn3Ca-cluster with a dangler Mn) to the ability to discriminate between different mechanistic proposals (e.g. showing that the Babcock scheme for water oxidation is unlikely).     

Characterization of the Streptomyces lividans PspA response

Phage shock protein (Psp) is induced by extracytoplasmic stress that may reduce the energy status of the cell. It is encoded in Escherichia coli by the phage shock protein regulon consisting of pspABCDE and by pspF and pspG. The phage shock protein system is highly conserved among a large number of gram-negative bacteria. However, many bacterial genomes contain only a pspA homologue but no homologues of the other genes of the Psp system. This conservation indicates that PspA alone might play an important role in these bacteria. In Streptomyces lividans, a soil-borne gram-positive bacterium, the phage shock protein system consists only of the pspA gene. In this report, we showed that pspA encodes a 28-kDa protein that is present in both the cytoplasmic and the membrane fractions of the S. lividans mycelium. We demonstrated that the pspA gene is strongly induced under stress conditions that attack membrane integrity and that it is essential for growth and survival under most of these conditions. The data reported here clearly show that PspA plays an important role in S. lividans under stress conditions despite the absence of other psp homologues, suggesting that PspA may be more important in most bacteria than previously thought.     

Control of Cell Proliferation,Organ Growth,and DNA Damage Response Operate Independently of Dephosphorylation of the Arabidopsis Cdk1 Homolog CDKA;1

Entry into mitosis is universally controlled by cyclin-dependent kinases (CDKs). A key regulatory event in metazoans and fission yeast is CDK activation by the removal of inhibitory phosphate groups in the ATP binding pocket catalyzed by Cdc25 phosphatases. In contrast with other multicellular organisms, we show here that in the flowering plant Arabidopsis thaliana, cell cycle control does not depend on sudden changes in the phosphorylation pattern of the PSTAIRE-containing Cdk1 homolog CDKA;1. Consistently, we found that neither mutants in a previously identified CDC25 candidate gene nor plants in which it is overexpressed display cell cycle defects. Inhibitory phosphorylation of CDKs is also the key event in metazoans to arrest cell cycle progression upon DNA damage. However, we show here that the DNA damage checkpoint in Arabidopsis can also operate independently of the phosphorylation of CDKA;1. These observations reveal a surprising degree of divergence in the circuitry of highly conserved core cell cycle regulators in multicellular organisms. Based on biomathematical simulations, we propose a plant-specific model of how progression through the cell cycle could be wired in Arabidopsis.     

Construction of a Gene Knockout System for Application in Paenibacillus alvei CCM 2051T,Exemplified by the S-Layer Glycan Biosynthesis Initiation Enzyme WsfP

The gram-positive bacterium Paenibacillus alvei CCM 2051^T is covered by an oblique surface layer (S-layer) composed of glycoprotein subunits. The S-layer O-glycan is a polymer of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that is linked by an adaptor of -[GroA-2→OPO₂→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d-Galp-(1→ to specific tyrosine residues of the S-layer protein. For elucidation of the mechanism governing S-layer glycan biosynthesis, a gene knockout system using bacterial mobile group II intron-mediated gene disruption was developed. The system is further based on the sgsE S-layer gene promoter of Geobacillus stearothermophilus NRS 2004/3a and on the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. As a target gene, wsfP, encoding a putative UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase, representing the predicted initiation enzyme of S-layer glycan biosynthesis, was disrupted. S-layer protein glycosylation was completely abolished in the insertional P. alvei CCM 2051^T wsfP mutant, according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis evidence and carbohydrate analysis. Glycosylation was fully restored by plasmid-based expression of wsfP in the glycan-deficient P. alvei mutant, confirming that WsfP initiates S-layer protein glycosylation. This is the first report on the successful genetic manipulation of bacterial S-layer protein glycosylation in vivo, including transformation of and heterologous gene expression and gene disruption in the model organism P. alvei CCM 2051^T.Bacterial cell surface layer (S-layer) glycoproteins provide a unique self-assembly matrix that has been optimized by nature for regular and periodic display of glycans with nanometer scale accuracy (21, 31). Exploitation of this self-assembly system for surface display of functional, tailor-made glycans is an attractive alternative to the use of common cell surface anchors (7), with potential areas of application relating to any biological phenomenon that is based on carbohydrate recognition, such as receptor-substrate interaction, signaling, or cell-cell communication. A prerequisite for this endeavor is the availability of an S-layer glycoprotein-covered bacterium that is amenable to genetic manipulation. Despite the high application potential offered by the S-layer glycan display system, there are so far only two reports in the literature dealing with the genetic manipulation of S-layer glycoprotein-carrying bacteria. Both reports concern the gram-negative periodontal pathogen Tannerella forsythia ATCC 43037, but neither of them affects S-layer protein glycosylation (12, 24). In archaea, in contrast, molecular studies of S-layer protein glycosylation are quite advanced (1), but with the archaeal system, S-layer glycoprotein self-assembly, which is a prerequisite for the desired glycan display, has not been manageable in vitro so far.Our model organisms and, hence, candidates for S-layer-mediated glycan display enabled by carbohydrate engineering techniques are members of the Bacillaceae family. Currently, the S-layer glycosylation system of the thermophilic bacterium Geobacillus stearothermophilus NRS 2004/3a is best understood (20, 23, 29, 33, 34). However, a drawback of this organism is its resistance to take up foreign DNA. Although described in the literature (13, 14, 37), transformation of thermophilic bacilli seems to be a strain-specific trait. Based on successful transformation experiments in our laboratory, the mesophilic bacterium Paenibacillus alvei CCM 2051^T (ATCC 6344; DSM 29) (formerly Bacillus alvei [4]) was chosen to set up a system for genetic manipulation. The bacterium is completely covered with an oblique S-layer lattice composed of glycoprotein species. Various aspects of its S-layer, including ultrastructural characterization (27), glycosylation analysis (2, 18), and glycan biosynthesis (11), have been investigated so far. The S-layer O-glycans are polymers of [→3)-β-d-Galp-(1[α-d-Glcp-(1→6)]→4)-β-d-ManpNAc-(1→] repeating units that are linked via the adaptor -[GroA-2→OPO₂→4-β-d-ManpNAc-(1→4)]→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→3)-β-d- Galp-(1→ to specific tyrosine residues (2, 18) of the S-layer protein SpaA (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"FJ751775","term_id":"225546017","term_text":"FJ751775"}}FJ751775).Due to the presence of an identical adaptor saccharide backbone in the S-layer glycan of G. stearothermophilus NRS 2004/3a (29), where its biosynthesis is initiated by the UDP-Gal:phosphoryl-polyprenol Gal-1-phosphate transferase WsaP (33), it was conceivable that a homologous enzyme would initiate S-layer glycosylation in P. alvei CCM 2051^T. Considering that the S-layer protein glycosylation machinery has been found to be encoded by S-layer glycosylation (slg) gene clusters (21), degenerate primers for the rml genes catalyzing the dTDP-l-Rha biosynthesis required for building up the adaptor saccharide of the P. alvei CCM 2051^T S-layer glycan were used to define a point of entry into the glycosylation locus (K. Zarschler, B. Janesch, P. Messner, and C. Schäffer, unpublished data). Chromosome walking revealed the existence of an slg gene cluster of about 24 kb, including an open reading frame (ORF) predicted to encode the initiation enzyme of S-layer protein glycosylation. The corresponding gene, named wsfP, served as a first target for the gene knockout system developed in the course of the present study. This target was chosen because loss of function would be easily screenable, resulting in an S-layer glycosylation-deficient mutant. The gene knockout system constructed for insertional inactivation of the chromosomal wsfP gene of P. alvei CCM 2051^T is based on the commercially available bacterial mobile group II intron Ll.LtrB of Lactococcus lactis, in combination with further components available in our laboratory, including the broad-host-range S-layer gene promoter of sgsE from G. stearothermophilus NRS 2004/3a (22) and the Geobacillus-Bacillus-Escherichia coli shuttle vector pNW33N. Bacterial mobile group II introns are retroelements inserted into specific DNA target sites at high frequency by use of the retrohoming mechanism, by which the excised intron lariat RNA is inserted directly into a DNA target site and is then reverse transcribed by the associated intron-encoded enzyme protein (6, 8, 17). Since the DNA target site is recognized primarily by base pairing of intron RNA, which can be modified, and a few intron-encoded-enzyme-protein recognition positions, these introns can be inserted efficiently into any specific DNA target (9, 15, 35, 40).The aim of this study is the development of a genetic tool for manipulation of S-layer protein glycosylation in P. alvei CCM 2051^T. For proof of concept, we specifically deal with (i) the construction of a broad-host-range gene knockout system based on the L. lactis Ll.LtrB intron; (ii) its modification for specific disruption of the wsfP gene on the P. alvei CCM 2051^T chromosome, encoding the putative initiation enzyme of S-layer glycan biosynthesis; and (iii) the reconstitution of enzyme activity by plasmid-based expression of wsfP and its predicted functional homologue wsaP from G. stearothermophilus NRS 2004/3a.     

Structure-based drug design identifies novel LPA3 antagonists

Compound 5 ([5-(3-nitrophenoxy)-1,3-dioxo-1,3-dihydro-2-isoindol-2-yl]acetic acid) was identified as a weak selective LPA₃ antagonist (IC₅₀ = 4504 nM) in a virtual screening effort to optimize a dual LPA_{2 and 3} antagonist. Structure-based drug design techniques were used to prioritize similarity search matches of compound 5. This strategy rapidly identified 10 novel antagonists. The two most efficacious compounds identified inhibit activation of the LPA₃ receptor by 200 nM LPA with IC₅₀ values of 752 nM and 2992 nM. These compounds additionally define changes to our previously reported pharmacophore that will improve its ability to identify more potent and selective LPA₃ receptor antagonists. The results of the combined computational and experimental screening are reported.