BackgroundRecurrent abdominal pain is a common and costly health‐care problem attributed, in part, to visceral hypersensitivity. Increasing evidence suggests that gut bacteria contribute to abdominal pain perception by modulating the microbiome‐gut‐brain axis. However, specific microbial signals remain poorly defined. γ‐aminobutyric acid (GABA) is a principal inhibitory neurotransmitter and a key regulator of abdominal and central pain perception from peripheral afferent neurons. Although gut bacteria are reported to produce GABA, it is not known whether the microbial‐derived neurotransmitter modulates abdominal pain.MethodsTo investigate the potential analgesic effects of microbial GABA, we performed daily oral administration of a specific Bifidobacterium strain (B. dentium ATCC 27678) in a rat fecal retention model of visceral hypersensitivity, and subsequently evaluated pain responses.Key ResultsWe demonstrate that commensal Bifidobacterium dentium produces GABA via enzymatic decarboxylation of glutamate by GadB. Daily oral administration of this specific Bifidobacterium (but not a gadB deficient) strain modulated sensory neuron activity in a rat fecal retention model of visceral hypersensitivity.Conclusions & InferencesThe functional significance of microbial‐derived GABA was demonstrated by gadB‐dependent desensitization of colonic afferents in a murine model of visceral hypersensitivity. Visceral pain modulation represents another potential health benefit attributed to bifidobacteria and other GABA‐producing species of the intestinal microbiome. Targeting GABAergic signals along this microbiome‐gut‐brain axis represents a new approach for the treatment of abdominal pain.

Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2−/−/MMP9−/− mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2−/−/MMP9−/− allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.

RPPA experimental Data and Matlab source codes of modularized factor graph for parameter estimation are freely available online at http://ctsb.is.wfubmc.edu/publications/modularized-factor-graph.php.

Eight analogues of human epidermal growth factor (hEGF) having specific amino acid substitutions in the beta-sheet structure (residues 19-31) of the amino-terminal domain were generated by site-directed mutagenesis. Affinity of the epidermal growth factor (EGF) receptor for each of these mutant hEGF analogues was measured by both radioreceptor competition binding and receptor tyrosine kinase stimulation assays. The relative binding affinities obtained by these two methods were generally in agreement for each hEGF species. The results indicate that hydrophobic residues on the exposed surface of the beta-sheet structure of the amino-terminal domain of hEGF have an important role in the formation of the active EGF-receptor complex. The substitution of hydrophobic amino acid residues, Val-19----Gly, Met-21----Thr, Ile-23----Thr, and Leu-26----Gly, resulted in decreased binding affinity, with the most severe reductions observed with the last two mutants. The mutations Ala-25----Val and Lys-28----Arg introduced amino acid residues resulting in slightly increased receptor binding affinity. Similar to previous results with acidic residues in this region [Engler, D.A., Matsunami, R.K., Campion, S.R., Stringer, C.D., Stevens, A., & Niyogi, S.K. (1988) J. Biol. Chem. 263, 12384-12390], removal of the positive charge in the Lys-28----Leu substitution had almost no effect on binding affinity, indicating the lack of any absolute requirement for ionic interactions at this site. Substitution of Tyr-22, which resulted in decreased receptor binding affinity, provides further indication of the importance of aromatic residues in this region of the molecule, as found earlier with Tyr-29 (cf. reference above).(ABSTRACT TRUNCATED AT 250 WORDS)

Current delivery platforms are typically designed for prolonged circulation that favors superior accumulation of the payload in the targeted tissue. The design of efficient surface modifications determines both a longer circulation time and targeting abilities of particles. The optimization of synthesis protocols to efficiently combine targeting molecules and elements that allow for an increased circulation time can be challenging and almost impossible when several functional elements are needed. On the other hand, in the last decade, the development of bioinspired technologies was proposed as a new approach with which to increase particle safety, biocompatibility and targeting, while maintaining the synthesis protocols simple and reproducible. Recently, we developed a new drug delivery system inspired by the biology of immune cells called leukolike vector (LLV) and formed by a nanoporous silicon core and a shell derived from the leucocyte cell membrane. The goal of this study is to investigate the protein content of the LLV. Here we report the proteomic profiling of the LLV and demonstrate that our approach can be used to modify the surface of synthetic particles with more than 150 leukocyte membrane-associated proteins that determine particle safety, circulation time and targeting abilities towards inflamed endothelium.

Seven site-specific mutants (including changes to other hydrophobic, charged, and heterocyclic amino acids) of leucine 47 of human epidermal growth factor (EGF) were generated by protein engineering and characterized for their activity in three assays: radioreceptor competition binding in membrane fractions, the stimulation of the EGF receptor's tyrosine kinase activity, and the stimulation of thymidine uptake in tissue culture cells. K1/2 (concentration required for half maximum response) values for each of the mutants are reported in the three assays. The results show that the native leucine residue is quite important for EGF activity. Substitutions are tolerated to different degrees, depending upon hydrophobicity and size of the side chain. Substitution with ionic residues led to the most drastic reduction in activity. One-dimensional nuclear magnetic resonance spectroscopy, at physiological pH, of several of the mutants did not detect any major structural perturbations which would account for the loss of activity. The results suggest that the side chain of leucine 47, because of its charge neutrality, size, and hydrophobicity, is highly important, although not absolutely essential for the interaction of EGF with its receptor. A striking finding was the lower (compared with wild type) Vmax values of the mutants in the tyrosine kinase reaction, but these low Vmax mutants, in cell culture experiments, were able to stimulate at high concentrations a growth response equivalent to wild type EGF.

Anion-exchange chromatography resolves human plasma low-density lipoprotein (LDL) into 5 subfractions, with increasing negative surface charge in the direction of L1 to L5. Unlike the harmless L1 to L4, the exclusively atherogenic L5 is rejected by the normal LDL receptor (LDLR) but endocytosed into vascular endothelial cells through the lectin-like oxidized LDL receptor-1 (LOX-1). Analysis with SDS-PAGE and 2-dimensional electrophoresis showed that the protein framework of L1 was composed mainly of apolipoprotein (apo) B100, with an isoelectric point (pI) of 6.620. There was a progressively increased association of additional proteins, including apoE (pI 5.5), apoAI (pI 5.4), apoCIII (pI 5.1), and apo(a) (pI 5.5), from L1 to L5. LC/MSE was used to quantify protein distribution in all subfractions. On the basis of weight percentages, L1 contained 99% apoB-100 and trace amounts of other proteins. In contrast, L5 contained 60% apoB100 and substantially increased amounts of apo(a), apoE, apoAI, and apoCIII. The compositional characteristics contribute to L5’s electronegativity, rendering it unrecognizable by LDLR. LOX-1, which has a high affinity for negatively charged ligands, is known to mediate the signaling of proinflammatory cytokines. Thus, the chemical composition–oriented receptor selectivity hinders normal metabolism of L5, enhancing its atherogenicity through abnormal receptors, such as LOX-1.

Three site-directed mutants of human epidermal growth factor, Leu-26+Gly, Leu-47-+Ala, and Be-23-+Thr, were examined for their ability to stimulate the protein-tyrosine kinase activity of the epidermal growth factor receptor. The receptor binding atiinities of the mutant growth factors were 20-to 50-fold lower, as compared to wild-type growth factor. At saturating concentrations of growth factor, the velocities of the phosphorylation of exogenously added substrate and receptor autophosphorylation were significantly lower with the mutant analogs, suggesting a partial 'uncoupling' of signal transduction. The mutant analogs were shown to compete directly with the binding of wild-type, resulting in a decrease in growth factor-stimulated kinase activity.Epidermal growth factor analog; Epidermal growth factor receptor kinase; Competitive inhibitors of epidermal growth factor