Mechanistic Insight into the TH1-Biased Immune Response to Recombinant Subunit Vaccines Delivered by Probiotic Bacteria-Derived Outer Membrane Vesicles

Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV’s ability to induce self-adjuvanted robust and protective humoral and T_H1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific T_H1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered “pathogen-like particles” derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.     

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.     

An exploration of the binding site of aldolase using alkyl glycolamido phosphoric esters and alkyl monoglycolate phosphoric esters.

Alkyl glycolamido phosphoric esters (P-O-CH2-CO-NH-(CH2)n-CH3) and alkyl monoglycolate phosphoric esters (P-O-CH2-CO-O-(CH2)n-CH3), which are analogs of the aldolase substrate fructose-1-phosphate, were synthesized and use for probing the active site of rabbit muscle aldolase. The inhibition constants (Ki) were affected by the length of the alkyl groups of these compounds and a maximum value of Ki was observed between the number of methylene groups 2 and 4, depending on the type of compound. In the previous investigation, N-(omega-hydroxyalkyl)-glycolamido bisphosphoric esters (P-O-CH2-CO-NH-(CH2)n-O-P) and alkanediol monoglyclolate bisphosphoric esters (P-O-CH2-CO-O-(CH2)n-O-P) have a minimum Ki value between the number of methylene groups 1 and 4. The difference spectra of aldolase caused by binding of alkyl glycoamido phosphoric esters or alkyl monophosphates resembled that of their analogous bisphosphoric esters, but the intensity of absorbance was smaller than that of the bisphosphoric ester analogs. These results suggest that rabbit muscle aldolase has two binding sites for the phosphate groups on the entrance end of the active site cavity, the singly wound beta-barrel of the parallel alpha/beta class structure. The distance between the phosphate binding site Lys-107 in the beta-sheet structure (c) and Arg-148 in the beta-sheet structure (d) may possibly be expanded or contracted by the forms of the bending structure of the biphosphate compounds. Also, the change of distance between the beta-sheet structure (c) and (d) containing Trp-147, may have an effect on the environment of the tryptophan and cause a change of the absorbance of aldolase especially at 295-299 nm. On the other hand, the synthetic monophosphate compounds bind at only one of the two phosphate binding sites and have very little effect on the absorbance of Trp-147, in a similar manner as orthophosphate. The alkyl groups of monophosphate may be repelled by the ionic amino acid side chains, Asp-33, Lys-146, Glu-187 and/or Lys-229 in the middle of the active site cavity. However, the end of the long alkyl group of some monophosphates may possibly contact the hydrophobic bottom of the active site cavity without effect on the environment of Trp-147.     

Coarse-grained molecular simulations of large biomolecules

Recently, we saw a dramatic increase in the number of researches that rely on coarse-grained (CG) simulations for large biomolecules. Here, first, we briefly describe recently developed and used CG models for proteins and nucleic acids. Balance between structure-based and physico-chemical terms is a key issue. We also discuss the multiscale algorithms used to derive CG parameters. Next, we comment on the dynamics used in CG simulations with an emphasis on the importance of hydrodynamic interactions. We then discuss the pros and cons of CG simulations. Finally, we overview recent exciting applications of CG simulations. Publicly available tools and software for CG simulations are also summarized.     

Cloning and expression of the isocitrate lyase gene from a nitrogen-fixing bacterium, Azotobacter vinelandii, and functional analysis of the enzyme by site-directed mutagenesis

The gene encoding isocitrate lyase (ICL) from a nitrogen-fixing mesophilic bacterium, Azotobacter vinelandii strain IAM1078, was cloned, and the gene expression was examined. When sodium acetate or glucose was used as carbon source, similar growth was observed in this bacterium, but the ICL activity of cells grown with the former source was 43-hold higher than those with the latter. In addition, northern blot analysis revealed that expression of the ICL gene was induced by acetate. Based on a comparison of the amino acid sequences of the ICLs of various organisms, the ICL of this bacterium was found to be classifiable into subfamily 3, one of two phylogenetic groups of eubacteial ICLs. Replacement of the Ile504 in the ICL by Met, which is conserved in the corresponding position of cold-adapted ICLs of psychrophlic bacteria, resulted in decreased thermostability of activity, indicating that this amino acid residue is involved in thermal properties of this enzyme.     

Acremomannolipin A,the potential calcium signal modulator with a characteristic glycolipid structure from the filamentous fungus Acremonium strictum

By the newly developed assay method, the glycolipid Acremomannolipin A (1) was isolated from a filamentous fungus Acremonium strictum as a potential calcium signal modulator. The structure of 1 elucidated on the basis of intensive spectroscopic analyses as well as its degradation studies is quite unique: the d-mannopyranose is connected to d-mannitol through a β-glycoside linkage; all the hydroxyls in the mannose are highly masked as peresters with aliphatic acids, and this moiety is made hydrophobic, whereas the mannitol part exhibits a highly hydrophilic property. The compound (1) showed the characteristic bioactivity property, enabling calcineurin deletion cells to grow in the presence of Cl^?, which would be caused by calcium signal modulating. The activity was so potent as to exert the effect at a concentration of 200 nM.     

Immunohistochemical investigation of lymphatic vessel formation control in mouse tooth development: lymphatic vessel-forming factors and receptors in tooth development in mice

The presence of lymphatic vessels in dental pulp has recently been controversial, and no conclusion has been reached. In this study, we investigated the control of lymphangiogenesis with dental pulp development in the mouse mandibular molar using VEGF-C, VEGF-D, and VEGFR-3 as indices of lymphatic vessel-controlling factors. In addition, to distinguish blood and lymphatic vascular epithelial cells, we performed immunohistochemical analysis using von Willebrand factor (vWF) and statistical analysis. In dental papilla in the bell-stage non-calcified period, mesenchymal cells positive for VEGF-C, VEGF-D, and VEGFR-3 increased and lumen-forming endothelial cells were noted, but vWF was negative, suggesting that these were actively forming lymphatic vessels. Positive undifferentiated mesenchymal cells, an increase in endothelial cells in dental pulp, and lumen expansion were noted early after birth. Positivity was also detected in the odontoblast layer and sheath of Hertwig after birth, suggesting that these factors also play important roles in odontoblast differentiation and maturation and periodontal ligament and tooth root formation. We embryologically clarified lymphatic vessel formation in dental pulp and a process of lymphatic vessel formation from blood vessels, suggesting involvement of the surrounding tissue, odontoblasts, and sheath of Hertwig in vessel formation.