Opr86 is essential for viability and is a potential candidate for a protective antigen against biofilm formation by Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that is one of the most refractory to therapy when it forms biofilms in the airways of cystic fibrosis patients. To date, studies regarding the production of an immunogenic and protective antigen to inhibit biofilm formation by P. aeruginosa have been superficial. The previously uncharacterized outer membrane protein (OMP) Opr86 (PA3648) of P. aeruginosa is a member of the Omp85 family, of which homologs have been found in all gram-negative bacteria. Here we verify the availability of Opr86 as a protective antigen to inhibit biofilm formation by P. aeruginosa PAO1 and several other isolates. A mutant was constructed in which Opr86 expression could be switched on or off through a tac promoter-controlled opr86 gene. The result, consistent with previous Omp85 studies, showed that Opr86 is essential for viability and plays a role in OMP assembly. Depletion of Opr86 resulted in streptococci-like morphological changes and liberation of excess membrane vesicles. A polyclonal antibody against Opr86 which showed reactivity to PAO1 cells was obtained. The antibody inhibited biofilm formation by PAO1 and the other clinical strains tested. Closer examination of early attachment revealed that cells treated with the antibody were unable to attach to the surface. Our data suggest that Opr86 is a critical OMP and a potential candidate as a protective antigen against biofilm formation by P. aeruginosa.     

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at −5 °C for 30 min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60 min at 25 °C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16 Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25 °C for 30 min, the maturation rate decreased in solution with 0.51 Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40 Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25 °C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at −5 °C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.     

Effect of Different Levels of Pressure Relieving Air-Mattress Firmness on Cough Strength

Cough is an important host-defense mechanism. The elderly and patients who are severely ill cannot cough effectively when lying in the supine position. Furthermore, pressure relieving air-mattresses are recommended for preventing the development of pressure ulcers. In this study, we clarified whether or not the cough peak flow (CPF), an index of cough strength, is affected by different firmness levels of a pressure relieving air-mattress in healthy volunteers in the supine position. Fifty-two healthy young men participated. All the measurements were carried out on each participant in the supine position on a pressure relieving air-mattress. The participants were assessed at two firmness levels, a “hard” and “soft” mode. The CPF, forced vital capacity (FVC), maximal expiratory pressure (PEmax), and maximal inspiratory pressure (PImax) were determined for each mode. The sinking distance of the body into the mattress was measured without any activity and the difference between the sinking distances of the two firmness levels was determined. The CPF, FVC, PEmax, and PImax were determined for each mode. The sinking distance of the body into the mattress was measured and the difference between the sinking distances of the two firmness levels was determined. The CPF, FVC, PEmax and PImax values of the participants coughing on the mattress were significantly lower when the mattress was in “soft” than in “hard” mode. The differences between the sinking distances of the mattress in “soft” and “hard” modes were larger for the anterior superior iliac spine. A harder mattress may lead to increased CPF in healthy young men lying in the supine position, and increased CPF may be important for host defense.     

Reduction of sooty mold damage through biocontrol of the greenhouse whitefly <Emphasis Type="Italic">Trialeurodes vaporariorum</Emphasis> (Hemiptera: Aleyrodidae) using selective insecticides in tomato cultivation greenhouses

The proliferation of sooty mold on tomato fruit, Solanum lycopersicum L.—as caused by the secretion of honeydew on the fruit by greenhouse whitefly, Trialeurodes vaporariorum (Westwood) (Hemiptera: Aleyrodidae)—has recently become a serious problem in estivo-autumnal greenhouse tomato cultivation in Japan. It is becoming increasingly difficult to control T. vaporariorum using insecticides because whiteflies have developed resistance to a variety of insecticides. As the first step towards integrated pest management, we examined whether the use of selective insecticides could prompt a cascade process in which an increase in parasitoids is followed by a decrease in whitefly occurrence and then a reduction in sooty mold damage. We compared greenhouses in which nonselective insecticides effective against T. vaporariorum and its parasitoids were used (hereafter denoted “nonselective insecticide greenhouses”) with greenhouses in which selective insecticides for T. vaporariorum were used (hereafter denoted “selective insecticide greenhouses”) in terms of the density and level of parasitism of T. vaporariorum as well as the degree of damage from sooty mold. The number of parasitized whiteflies increased with the number of whiteflies in the selective insecticide greenhouses, whereas it remained at low levels regardless of the number of whiteflies in the nonselective insecticide greenhouses. Furthermore, the selective insecticide greenhouses showed significantly higher parasitism levels, fewer whiteflies, and reduced sooty mold damage compared to the nonselective insecticide greenhouses. These results suggest that the use of selective insecticides causes an increase in parasitism, which in turn suppresses the number of greenhouse whiteflies and, eventually, sooty mold.     

Design,synthesis, and biological evaluation of novel biphenyl-4-carboxamide derivatives as orally available TRPV1 antagonists

A new series of transient receptor potential vanilloid type 1 (TRPV1) antagonists were designed and synthesized from N-(3-hydroxyphenyl)-2-(piperidin-1-ylmethyl)biphenyl-4-carboxamide hydrochloride (8). SAR studies identified (R)-N-(1-methyl-2-oxo-1,2,3,4-tetrahydro-7-quinolyl)-2-[(2-methylpyrrolidin-1-yl)methyl]biphenyl-4-carboxamide hydrochloride (ASP8370, 7), as a compound with high aqueous solubility, satisfactory stability in human liver microsomes, and reduced CYP3A4 inhibition. ASP8370 was selected as a clinical development candidate with significant ameliorative effects on neuropathic pain. SAR studies also revealed the structural mechanisms underlying the switching between TRPV1 antagonism and agonism.     

Effect of Inorganic Orthophosphate on in Vitro Activity of NADH-Nitrate Reductase Isolated from 2-Row Barley Leaves

Inorganic orthophosphate (25 millimolar in assay media; Pi) was found to increase in vitro activity of NADH-nitrate reductase (NR) isolated from 2-row barley (Hordeum vulgare L.) leaves with a saturating concentration of nitrate (2 millimolar) but to decrease it with low nitrate levels (<0.1 millimolar). The response to nitrate concentrations was Pi specific. The Lineweaver-Burk plot showed that Pi increases the apparent K_m for nitrate as well as V_max, whereas it does not alter the K_m for NADH significantly. These results suggest that the interaction between a molybdenum site of the enzyme and Pi results in alteration of the properties of NR molecule.     

Sequential expansion of antibody heterogeneity during the response to bacterial alpha-amylase

By analyzing antibody heterogeneity during the primary immune response to bacterial a-amylase (B alpha A) in high-responder F1 hybrid mice between C57BL/6 (B6) and C3H/He (C3) mice with the use of isoelectric focusing (IEF), it was shown that the maturation of the primary IgG antibody response consisted of at least two stages. The response of every mouse tested was initiated with the production of specific antibody focused as a limited set of bands in a narrow pH range, and the subsequent rise in antibody titer was associated with the sequential expansion of the spectra involving the appearance of new bands in the pH gradient adjacent to the initial bands. A further rise was accompanied only by intensified staining of the pre-existing bands. These two stages were distinguishable regardless of the antigen dose, although increasing the dose led to widely distributed spectra of focused antibodies and an early shift from the first stage to the second. The sequential expansion of spectra following the appearance of initial antibodies with limited isoelectric point (pI) values was not unique to the anti-BaA antibody response, because similar results were obtained with the antibody response to an immunologically unrelated antigen, Taka-amylase A (TAA). Thus, the appearance of initial antibodies in a limited pH range, overlapping among all F1 hybrids tested, is not a direct reflection of similarity in the determinant specificities of these antibodies among different mice.     

Mechanical characterization of adult stem cells from bone marrow and perivascular niches

Therapies using adult stem cells often require mechanical manipulation such as injection or incorporation into scaffolds. However, force-induced rupture and mechanosensitivity of cells during manipulation is largely ignored. Here, we image cell mechanical structures and perform a biophysical characterization of three different types of human adult stem cells: bone marrow CD34+ hematopoietic, bone marrow mesenchymal and perivascular mesenchymal stem cells. We use micropipette aspiration to characterize cell mechanics and quantify deformation of subcellular structures under force and its contribution to global cell deformation. Our results suggest that CD34+ cells are mechanically suitable for injection systems since cells transition from solid- to fluid-like at constant aspiration pressure, probably due to a poorly developed actin cytoskeleton. Conversely, mesenchymal stem cells from the bone marrow and perivascular niches are more suitable for seeding into biomaterial scaffolds since they are mechanically robust and have developed cytoskeletal structures that may allow cellular stable attachment and motility through solid porous environments. Among these, perivascular stem cells cultured in 6% oxygen show a developed cytoskeleton but a more compliant nucleus, which can facilitate the penetration into pores of tissues or scaffolds. We confirm the relevance of our measurements using cell motility and migration assays and measure survival of injected cells. Since different types of adult stem cells can be used for similar applications, we suggest considering mechanical properties of stem cells to match optimal mechanical characteristics of therapies.