Effect of pore architecture on oxygen diffusion in 3D scaffolds for tissue engineering

The aim of this study was to maximize oxygen diffusion within a three-dimensional scaffold in order to improve cell viability and proliferation. To evaluate the effect of pore architecture on oxygen diffusion, we designed a regular channel shape with uniform diameter, referred to as cylinder shaped, and a new channel shape with a channel diameter gradient, referred to as cone shaped. A numerical analysis predicted higher oxygen concentration in the cone-shaped channels than in the cylinder-shaped channels, throughout the scaffold. To confirm these numerical results, we examined cell proliferation and viability in 2D constructs and 3D scaffolds. Cell culture experiments revealed that cell proliferation and viability were superior in the constructs and scaffolds with cone-shaped channels.     

Development of a noncompetitive phage anti-immunocomplex assay for brominated diphenyl ether 47

We present a new application of the noncompetitive phage anti-immunocomplex assay (PHAIA) by converting an existing competitive assay to a versatile noncompetitive sandwich-type format using immunocomplex binding phage-borne peptides to detect the brominated flame retardant, brominated diphenyl ether 47 (BDE 47). Three phage-displayed 9-mer disulfide-constrained peptides that recognize the BDE 47-polyclonal antibody immunocomplex were isolated. The resulting PHAIAs showed variable sensitivities, and the most sensitive peptide had a dose-response curve with an SC₅₀ (concentration of analyte producing 50% saturation of the signal) of 0.7 ng/ml BDE 47 and a linear range of 0.3-2 ng/ml, which was nearly identical to the best heterologous competitive format (IC₅₀ of 1.8 ng/ml, linear range of 0.4-8.5/ml). However, the PHAIA was 1400-fold better than homologous competitive assay. The validation of the PHAIA with extracts of house furniture foam as well as human and calf sera spiked with BDE 47 showed overall recovery of 80-113%. The PHAIA was adapted to a dipstick format (limit of detection of 3.0 ng/ml), and a blind test with six random extracts of local house furniture foams showed that the results of the PHAIA and dipstick assay were consistent, giving the same positive and negative detection.     

Therapeutic effect of (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) against Staphylococcus aureus infection in a murine model

Sortase enzymes belong to a family of transpeptidases found in Gram-positive bacteria. Sortase is responsible for the reaction that anchors surface protein virulence factors to the peptidoglycan cell wall of the bacteria. The compound (Z)-3-(2,5-dimethoxyphenyl)-2-(4-methoxyphenyl) acrylonitrile (DMMA) has previously been reported as a novel sortase inhibitor in vitro, but the in vivo effects of DMMA have not been studied. Here, we evaluated the in vivo effects of DMMA against infection by wild-type and sortase A- and/or sortase B-deficient Staphylococcus aureus in Balb/c mice. With DMMA treatment, survival rates increased and kidney and joint infection rates decreased (p < 0.01) in a dose-dependent manner. The rate of kidney infection was significantly reduced in the mice treated with sortase A knock-out S. aureus (p < 0.01). These results indicate that by acting as a potent inhibitor of sortase A and moderate inhibitor of sortase B, DMMA can decrease kidney and joint infection rates and reduce mortality in mice infected with S. aureus. These findings suggest that DMMA is a promising therapeutic compound against Gram-positive bacteria.     

Eisenstasin,new antistasin family inhibitor from the earthworm

A couple of new antistasin family serine protease inhibitors have been isolated from the non-hematophagous earthworm, Eisenia andrei. These novel inhibitors have been designated as eisenstasin I and II. Similar to other antistasin family inhibitors, eisenstasin I and II feature 3 and 4 internal repeats, respectively, of a 24–29 amino acid sequence, both of which exhibit a conserved pattern of 6-cysteine/2-glycine at an identical position between the third and fourth cysteine residues. This suggests that the eisenstasins isolated from the earthworm are members of the antistasin family. The eisenstasins are 82% similar with regard to amino acid sequences and exhibit over 70% similarity with the antistasins from the earthworm Lumbricus rubellus, while also displaying less than 40% sequence similarity with the leech antistasins. Earthworm eisenstasins are basic proteins, primarily due to the frequent occurrence of arginine residues in their structure, especially at the C-terminal region. As arginine is a key residue for the substrate specificity of some serine proteases including FXa, it is thought that these multiple arginine residues may play a role in the inhibitory characteristics of the eisenstasins. Considering the structure and number of the internal repeats derived from a variety of animal species, the deletion as well as the duplication of all or part of an internal repeat may be implicated in the evolution of the structure and function of the antistasin family inhibitors.     

Production of human CD59-transgenic pigs by embryonic germ cell nuclear transfer

This study was performed to produce transgenic pigs expressing the human complement regulatory protein CD59 (hCD59) using the nuclear transfer (NT) of embryonic germ (EG) cells, which are undifferentiated stem cells derived from primordial germ cells. Because EG cells can be cultured indefinitely in an undifferentiated state, they may provide an inexhaustible source of nuclear donor cells for NT to produce transgenic pigs. A total of 1980 NT embryos derived from hCD59-transgenic EG cells were transferred to ten recipients, resulting in the birth of fifteen piglets from three pregnancies. Among these offspring, ten were alive without overt health problems. Based on PCR analysis, all fifteen piglets were confirmed as hCD59 transgenic. The expression of the hCD59 transgene in the ten living piglets was verified by RT-PCR. Western analysis showed the expression of the hCD59 protein in four of the ten RT-PCR-positive piglets. These results demonstrate that hCD59-transgenic pigs could effectively be produced by EG cell NT and that such transgenic pigs may be used as organ donors in pig-to-human xenotransplantation.     

Classification of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>l. japonica nipponbare) immunophilins (FKBPs,CYPs) and expression patterns under water stress

Background  

FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are abundant and ubiquitous proteins belonging to the peptidyl-prolyl cis/trans isomerase (PPIase) superfamily, which regulate much of metabolism through a chaperone or an isomerization of proline residues during protein folding. They are collectively referred to as immunophilin (IMM), being present in almost all cellular organs. In particular, a number of IMMs relate to environmental stresses.     

Genome-scale metabolic reconstruction and <Emphasis Type="Italic">in silico</Emphasis> analysis of methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis> for strain improvement

Background  

Pichia pastoris has been recognized as an effective host for recombinant protein production. A number of studies have been reported for improving this expression system. However, its physiology and cellular metabolism still remained largely uncharacterized. Thus, it is highly desirable to establish a systems biotechnological framework, in which a comprehensive in silico model of P. pastoris can be employed together with high throughput experimental data analysis, for better understanding of the methylotrophic yeast's metabolism.