Dichlorotriazine-based multivalent probe for selective affinity labeling of carbohydrate-binding proteins.

A new series of multivalent gold nanoparticle probes bearing different electrophilic groups were synthesized and their affinity labeling reactivities were evaluated. The dichlorotriazine group was identified as a useful protein-reactive label, allowing selective capture of a target protein at nanomolar probe concentrations.

Synthesis of Interlocked and Non-Interlocked Deca(para-phenylene) Derivatives by Ni-mediated Biaryl Coupling.

Oligo(para-phenylene) (PPn) is a compound composed of directly connected 1,4-phenylene moieties. The synthesis of PPn composed of six or more phenylene moieties with no substituent at the internal phenylene moiety has been challenging because of its low solubility. Herein we synthesized oligo(para-phenylene)[2]rotaxanes, including a deca(para-phenylene)[2]rotaxane, with a defined number of phenylene moieties. Biaryl coupling of iodoarenes mediated by macrocyclic dibenzodihydrophenanthroline-Ni complex was utilized for the first time to synthesize the [2]rotaxanes. Compared to the non-interlocked deca(para-phenylene), the deca(para-phenylene)[2]rotaxane showed higher solubility. The properties of the oligo(para-phenylene)[2]rotaxanes and non-interlocked oligo(para-phenylene)s were analyzed by spectroscopic methods.

Conformational Control of [2]Rotaxane by Hydrogen Bond.

A series of [2]rotaxanes with various functional groups in the axle component was synthesized by the oxidative dimerization of alkynes, which is mediated by a macrocyclic phenanthroline-Cu complex. The rotaxanes were fully characterized by spectroscopic methods, and the structure of a rotaxane was determined by X-ray crystallographic analysis. The interaction between the ring component and the axle component was studied in detail to understand the conformation of the rotaxanes. The presence of the hydrogen bond between the phenanthroline moiety in the macrocyclic component and the acidic proton in the axle component influenced the conformation of rotaxane.

CXCL1-Triggered PAD4 Cytoplasmic Translocation Enhances Neutrophil Adhesion through Citrullination of PDIA1.

AIMS: Vascular inflammation is critical for the development and progression of atherosclerosis. Previously, we reported that neutrophils adhere to the vascular endothelium in low-density lipoprotein receptor null mice fed a high-fat diet through hypercitrullination of histone H3 by peptidylarginine deiminase 4 (PAD4) in neutrophils. However, the involvement of PAD4 and citrullination of proteins other than histone H3 in neutrophil adhesion is not well known. In this study, we investigated the function of PAD4 and identified citrullinated proteins during vascular inflammation. METHODS: We pefformed flow assay under physiological flow conditions using differentiated HL-60 (dHL-60) cells stimulated with CXCL1 and human umbilical vein endothelial cells (HUVECs). Furthermore, phalloidin stain for dHL-60 stimulated with CXCL1 to observe F-actin polymerization and immunohistochemistry for the activated ß2-integrin was conducted. To identify a target of citrullination in the cytoplasm of dHL-60 cells, liquid chromatography-mass spectrometry (LC-MS/MS) for dHL-60 stimulated with CXCL1 was performed. RESULTS: Inhibition or knockdown of PAD4 significantly decreased adhesion of under physiological flow conditions. Thr-Asp-F-amidine trifluoroacetate salt (TDFA), a PAD4 inhibitor, inhibited cytoplasmic translocation of PAD4 by CXCL1. TDFA or knockdown of PAD4 significantly decreased expression of ß2-integrin and F-actin polymerization activated by CXCL1. Moreover, LC-MS/MS identified protein disulfide isomerase A1 (PDIA1) as a target of citrullination in the cytoplasm of dHL-60 cells. Knockdown of PDIA1 significantly decreased adhesion of dHL-60 cells to HUVECs, expression of ß2-integrin, and F-actin polymerization. CONCLUSIONS: Cytoplasmic translocation of PAD4 by CXCL1 induces neutrophil adhesion to vascular endothelial cells and citrullination of PDIA1.

Exploration of the Reactivity of Multivalent Electrophiles for Affinity Labeling: Sulfonyl Fluoride as a Highly Efficient and Selective Label.

Here we explored the reactivity of a set of multivalent electrophiles cofunctionalized with a carbohydrate ligand on gold nanoparticles to achieve efficient affinity labeling for target protein analysis. Evaluation of the reactivity and selectivity of the electrophiles against three different cognate binding proteins identified arylsulfonyl fluoride as the most efficient protein-reactive group in this study. We demonstrated that multivalent arylsulfonyl fluoride probe 4 at 50 nm concentration achieved selective affinity labeling and enrichment of a model protein PNA in cell lysate, which was more effective than photoaffinity probe 1 with arylazide group. Labeling site analysis by LC-MS/MS revealed that the nanoparticle-immobilized arylsulfonyl fluoride group can target multiple amino acid residues around the ligand binding site of the target proteins. Our study highlights the utility of arylsulfonyl fluoride as a highly effective multivalent affinity label suitable for covalently capturing unknown target proteins.

Synthesis of Mechanically Planar Chiral rac-[2]Rotaxanes by Partitioning of an Achiral [2]Rotaxane: Stereoinversion Induced by Shuttling.

Mechanically planar chiral [2]rotaxanes were synthesized by the introduction of bulky pyrrole moieties into the axle component of an achiral [2]rotaxane. The enantiomers were separated by chiral HPLC. The shuttling of the ring component between the two compartments at high temperature induced the stereoinversion of the mechanically planar chiral [2]rotaxane. The rate of the stereoinversion was studied quantitatively, and the kinetic parameters were determined.

Total synthesis of elastin peptide using high pressure-liquid phase synthesis assisted by a soluble tag strategy.

A highly aggregating elastin peptide was prepared efficiently using a high pressure-liquid phase synthesis approach assisted by a soluble tag strategy. Two standard syringes were connected to each other to construct a reactor. This simple reactor was used to apply high pressure to the highly viscous reaction mixture thereby maintaining its fluidity. The reactions were completely inhibited due to aggregation when conducted in a standard flask reactor, whereas our high pressure approach accelerated the couplings to realize complete conversion within 5-7 min. All steps were conducted at 0.10 M concentration, affording grams of the desired product.

Immunobiotic Lactobacillus jensenii as immune-health promoting factor to improve growth performance and productivity in post-weaning pigs.

BACKGROUND: Immunoregulatory probiotics (immunobiotics) have been proposed to improve piglets' immune system to avoid intestinal infections and reduce unproductive inflammation after weaning. Previously, it was demonstrated that Lactobacillus jensenii TL2937 (LjTL2937) attenuated the inflammatory response triggered by activation of Toll-like receptor 4 (TLR-4) in porcine intestinal epithelial (PIE) cells and antigen presenting cells (APCs) from porcine Peyer's patches (PP). OBJECTIVE: In view of the critical importance of PIE-APCs interactions in the regulation of intestinal immune responses, we aimed to examine the effect of LjTL2937 on activation patterns of APCs from swine PPs in co-cultures with PIE cells. In addition, we investigated whether LjTL2937 was able to beneficially modulate intestinal immunity of piglets after weaning to improve immune-health status. RESULTS: Stimulation of PIE-APCs co-cultures with LjTL2937 increased the expression of MHC-II, CD80/86, IL-10, and Bcl-3 in CD172a+CD11R1- and CD172a+CD11R1high APCs. In addition, the TL2937 strain caused the upregulation of three negative regulators of TLR4 in PIE cells: MKP-1, Bcl-3 and A20. These changes significantly reduced the inflammatory response triggered by TLR4 activation in PIE-APCs co-cultures. The in vivo experiments using castrated male piglets (crossbreeding (LWD) with Landrace (L), Large Yorkshire (W) and Duroc (D))of 3 weeks of age demonstrated that feeding with LjTL2937 significantly reduced blood complement activity and C reactive protein concentrations while no changes were observed in blood leukocytes, ratio of granulocytes to lymphocyte numbers, macrophages' activity and antibody levels. In addition, treatment with LjTL2937 significantly improved growth performance and productivity, and increased carcass quality. CONCLUSIONS: We demonstrated that the use of immunobiotics strains like LjTL2937, as supplemental additives for piglets feedings, could be used as a strategy to maintain and improve intestinal homeostasis; that is important for the development of the pig and for health and performance throughout the productive life of the animal.

Advanced application of porcine intestinal epithelial cells for the selection of immunobiotics modulating toll-like receptor 3-mediated inflammation.

PURPOSE: In this study, we aimed to characterize toll-like receptor (TLR)-3-mediated inflammatory immune response in porcine intestinal epithelial (PIE) cells and in PIE-immune cell co-cultures and, to evaluate if these in vitro systems are useful for selecting immunomodulatory lactic acid bacteria. RESULTS: We demonstrated that these systems are valuable tools for the in vitro study of the inflammatory response triggered by TLR3 in intestinal epithelial cells (IECs) and of the interaction between IECs and immune cells. In addition, we showed that PIE cells could be used for the selection of immunobiotic lactobacilli strains with anti-inflammatory activities. We found that Lactobacillus casei MEP221114 is an immunobiotic candidate for modulation of TLR3-mediated inflammatory responses. CONCLUSION: The present study deepened our understanding of the mechanisms of immunobiotic action by demonstrating that the interaction between some lactobacilli strains and IECs can up-regulate the mRNA expression of TLR negative regulators and that this effect could help to regulate the production of inflammatory mediators during the generation of a TLR3-mediated immune response.

Structural modulation and phase transitions in La2CoO(4.14) investigated by synchrotron X-ray and neutron single-crystal diffraction.

We report a combined synchrotron X-ray and neutron diffraction study on as-grown La(2)CoO(4.14) single-crystal from 10 to 470 K. Unprecedented structural features in terms of a (3 + 2)D incommensurate modulation have been detected and characterized in the Low Temperature Orthorhombic (LTO) phase already at room temperature despite the complex twinning that was unravelled. A new intermediate phase between the LTO and High Temperature Tetragonal (HTT) phases has been observed for the first time (in the range of 413-433 K). The transformation from LTO to this so-called HTLO (High Temperature Less Orthorhombic) phase is associated to a lowering of orthorhombicity and a loss of one modulation vector, yielding a (3 + 1)D incommensurate modulation. Conversely, above 433 K the HTT phase appears as nonmodulated but exhibits a strong dynamic disorder of CoO(6) octahedra, which has been characterized in detail by reconstruction of nuclear densities via the Maximum Entropy Method (MEM).

A newly established bovine intestinal epithelial cell line is effective for in vitro screening of potential antiviral immunobiotic microorganisms for cattle.

We evaluated whether a bovine intestinal epithelial (BIE) cell line could serve as a useful in vitro model system for studying antiviral immune responses in bovine intestinal epithelial cells (IECs) and for the primary screening of immunobiotic microorganisms with antiviral protective capabilities. Immunofluorescent analyses revealed that toll-like receptor 3 (TLR3) was expressed in BIE cells, and the results of real-time quantitative PCR showed that these cells respond to stimulation with poly(I:C) by up-regulating pro-inflammatory cytokines and type I interferons. In addition, we demonstrated that BIE cells are useful for the primary screening of immunobiotic lactic acid bacteria strains which are able to beneficially modulate antiviral immune responses triggered by TLR3 activation in bovine IECs. The characterization of BIE cells performed in the present study represents an important step towards the establishment of a valuable bovine in vitro system that could be used for the development of immunomodulatory feed for bovine hosts.

Immunobiotic Lactobacillus jensenii elicits anti-inflammatory activity in porcine intestinal epithelial cells by modulating negative regulators of the Toll-like receptor signaling pathway.

The effect of Lactobacillus jensenii TL2937 on the inflammatory immune response triggered by enterotoxigenic Escherichia coli (ETEC) and lipopolysaccharide (LPS) in a porcine intestinal epitheliocyte cell line (PIE cells) was evaluated. Challenges with ETEC or LPS elicited Toll-like receptor 4 (TLR4)-mediated inflammatory responses in cultured PIE cells, indicating that our cell line may be useful for studying inflammation in the guts of weaning piglets. In addition, we demonstrated that L. jensenii TL2937 attenuated the expression of proinflammatory cytokines and chemokines caused by ETEC or LPS challenge by downregulating TLR4-dependent nuclear factorκB (NF-κB) and mitogen-activated protein kinase (MAPK) activation. Furthermore, we demonstrated that L. jensenii TL2937 stimulation of PIE cells upregulated three negative regulators of TLRs: A20, Bcl-3, and MKP-1, deepening the understanding of an immunobiotic mechanism of action. L. jensenii TL2937-mediated induction of negative regulators of TLRs would have a substantial physiological impact on homeostasis in PIE cells, because excessive TLR inflammatory signaling would be downregulated. These results indicated that PIE cells can be used to study the mechanisms involved in the protective activity of immunobiotics against intestinal inflammatory damage and may provide useful information for the development of new immunologically functional feeds that help to prevent inflammatory intestinal disorders, including weaning-associated intestinal inflammation.

Immunobiotic lactic acid bacteria beneficially regulate immune response triggered by poly(I:C) in porcine intestinal epithelial cells.

This study analyzed the functional expression of TLR3 in various gastrointestinal tissues from adult swine and shows that TLR3 is expressed preferentially in intestinal epithelial cells (IEC), CD172a(+)CD11R1(high) and CD4(+) cells from ileal Peyer's patches. We characterized the inflammatory immune response triggered by TLR3 activation in a clonal porcine intestinal epitheliocyte cell line (PIE cells) and in PIE-immune cell co-cultures, and demonstrated that these systems are valuable tools to study in vitro the immune response triggered by TLR3 on IEC and the interaction between IEC and immune cells. In addition, we selected an immunobiotic lactic acid bacteria strain, Lactobacillus casei MEP221106, able to beneficially regulate the anti-viral immune response triggered by poly(I:C) stimulation in PIE cells. Moreover, we deepened our understanding of the possible mechanisms of immunobiotic action by demonstrating that L. casei MEP221106 modulates the interaction between IEC and immune cells during the generation of a TLR3-mediated immune response.

Psychromonas agarivorans sp. nov., a novel agarolytic bacterium.

Two heterotrophic, agarolytic bacteria were isolated from marine environments. A 16S rRNA gene sequence analysis showed the isolates (designated strains J42-3A(T) and 04OZ-AS15-7A) to be positioned in a separate lineage within the genus Psychromonas. Members of the genus Psychromonas are recognized as being psychrophilic or psychrotolerant, whereas none of the currently established Psychromonas species is known to be agarolytic. The DNA G+C contents of the isolates were about 42 mol% and the predominant cellular fatty acids were 16 : 1omega7c and 16 : 0. Based on the results of the phylogenetic and phenotypic analyses and DNA-DNA hybridization data, the isolates represent a novel species, for which the name Psychromonas agarivorans sp. nov. is proposed. The type strain is J42-3A(T) (=NBRC 104585(T)=KCTC 22285(T)).

Thalassomonas actiniarum sp. nov. and Thalassomonas haliotis sp. nov., isolated from marine animals.

Two marine heterotrophic bacteria, A5K-61(T) and A5K-106(T), were isolated from marine animals. 16S rRNA gene sequence analysis data showed that the isolates were affiliated with the genus Thalassomonas; highest 16S rRNA gene sequence similarity values were found with Thalassomonas viridans DSM 13754(T) (97.5 and 98.1 %, respectively). DNA-DNA hybridization values of strains A5K-61(T) and A5K-106(T) with T. viridans DSM 13754(T) (22.2-49.1 %) were clearly below 70 %, the generally accepted limit for species delineation. The isolates produced a brown diffusible pigment. The major respiratory quinone was Q-8 and the predominant cellular fatty acids were C(16 : 1)omega7c and C(16 : 0). Based on DNA-DNA hybridization data, some biochemical characteristics and 16S rRNA gene sequence comparison, the isolates represent two novel species of the genus Thalassomonas, for which the names Thalassomonas actiniarum sp. nov. (type strain A5K-106(T) =MBIC08328(T) =NCIMB 14418(T) =NBRC 104231(T)) and Thalassomonas haliotis sp. nov. (type strain A5K-61(T) =MBIC08329(T) =NCIMB 14417(T) =NBRC 104232(T)) are proposed.

Paramoritella alkaliphila gen. nov., sp. nov., a member of the family Moritellaceae isolated in the Republic of Palau.

Two strains of marine, heterotrophic, alkaliphilic bacteria, designated A3F-7(T) and ssthio04PA2-7c, were isolated from hard coral and marine sand, respectively, collected in the Republic of Palau. A phylogenetic analysis based on 16S rRNA gene sequences showed that the isolates were related to members of the genus Moritella. However, the sequence similarities between the isolates and the type strains of the Moritella species were less than 93 %. The G+C contents of the isolates were around 57 mol%, the major respiratory quinone was Q-8 and the predominant cellular fatty acids were 16 : 1omega7c, 16 : 0, 18 : 1omega7c and 14 : 0. On the basis of the phylogenetic data, phenotypic characteristics and DNA-DNA hybridization results, strains A3F-7(T) and ssthio04PA2-7c represent a novel species of a novel genus, for which the name Paramoritella alkaliphila gen. nov., sp. nov. is proposed. The type strain of Paramoritella alkaliphila is A3F-7(T) (=MBIC06429(T) =DSM 19956(T)).

Psychromonas heitensis sp. nov., a psychrotolerant bacterium isolated from seawater in Japan.

Four bacterial strains, 04HE-4-40, A4I-9, A4I-21 and AK15-027(T), were isolated from Heita Bay off Kamaishi in Japan. Based on 16S rRNA gene sequence analysis, the isolates were placed as a new lineage within the genus Psychromonas. The 16S rRNA gene sequence similarity values between the isolates and type strains of recognized Psychromonas species were less than 97 %. The DNA G+C contents of the isolates were about 38 mol%. The major respiratory quinone was Q-8, and the predominant cellular fatty acids were 16 : 1omega7c and 16 : 0. The isolates were able to grow at 30 degrees C, unlike other Psychromonas species. Based on the results of the phylogenetic analysis and phenotypic characteristics and DNA-DNA hybridization data, the isolates represent a novel species, for which the name Psychromonas heitensis sp. nov. is proposed. The type strain is AK15-027(T) (=MBIC 06898(T)=NCIMB 14416(T)).

Multilocus sequence typing and phylogenetic analyses of Pseudomonas aeruginosa Isolates from the ocean.

Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes.

Rapidithrix thailandica gen. nov., sp. nov., a marine gliding bacterium isolated from samples collected from the Andaman sea, along the southern coastline of Thailand.

The taxonomic positions of three strains of marine gliding bacteria, TISTR 1736, TISTR 1741 and TISTR 1750(T), isolated from the southern coastline of Thailand were evaluated by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the three isolates formed a distinct lineage within the family 'Flammeovirgaceae', phylum Bacteroidetes, and were related to the genus Flexithrix. The DNA G+C contents of the isolates were in the range 40-43 mol%. The major respiratory quinone was MK-7. The major cellular fatty acids were 16 : 1omega5c (cis-5-hexadecenoic acid) and 15 : 0 (pentadecanoic acid). The major hydroxyl fatty acids were 3-OH 17 : 0 (3-hydroxyheptadecanoic acid), 3-OH 15 : 0 (3-hydroxypentadecanoic acid) and 3-OH 16 : 0 (3-hydroxyhexadecanoic acid). On the basis of phenotypic, chemotaxonomic, genotypic and phylogenetic data, these marine bacteria are considered to represent a novel species of a new genus, for which the name Rapidithrix thailandica gen. nov., sp. nov. is proposed. The type strain of Rapidithrix thailandica is TISTR 1750(T) (=IAM 15448(T)).

Aureispira maritima sp. nov., isolated from marine barnacle debris.

A novel gliding marine bacterium (strain 59SA(T)) was isolated from marine barnacle debris. A phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate formed a distinct lineage within the genus Aureispira in the family 'Saprospiraceae'. The DNA G+C content of strain 59SA(T) was 38.7 mol%, the major respiratory quinone was MK-7 and the predominant cellular fatty acids were 20 : 4omega6c and 16 : 0. On the basis of the data from DNA-DNA hybridization, physiological and chemotaxonomic analyses and 16S rRNA gene sequence comparisons, strain 59SA(T) represents a novel species of the genus Aureispira, for which the name Aureispira maritima sp. nov. is proposed. The type strain is 59SA(T) (=IAM 15439(T)=TISTR 1726(T)).