Advanced Life Science Institute

The detection of mechanoradicals generated by homolytic bond scission enables the quantitative determination of the polymer network rupture during deformation in hydrogels. In this study, we investigate, in double network (DN) hydrogels, how the stretchable second network affects the stress distribution and rupture of the first network and how it contributes to energy dissipation by correlating bond scission with mechanical hysteresis. By increasing the second network density while keeping the first network constant, the tensile behavior of DN gels transitions from typical DN characteristics, exhibiting yielding with necking and strain-hardening, to an oversupported state, showing yielding and strain-hardening without necking, accompanied by reduced bond scission in the first network. Notably, the first network bond scission occurs extensively beyond the yield point and saturates at large strains, irrespective of necking behavior. Furthermore, the energy dissipated per bond cleavage remains constant in the necking regime but increases significantly in the strain-hardening regime and rises with higher second network density, indicating dissipation in the second network through internetwork entanglements. These trends are consistent across DN gels with both weak and strong cross-linkers in the first network, with the weak cross-linker leading to greater bond scission. This study not only deepens the understanding of the molecular mechanism underlying DN materials but also assists in designing mechanoresponsive functional materials based on DN mechanochemistry.

Polyelectrolyte complexation between oppositely charged polyelectrolytes forms coacervates in dilute solutions and thin films in concentrated solutions. It is difficult to obtain macroscopically uniform bulk polyelectrolyte complex (PEC) materials, since the two polymers form insoluble complexes quickly at the contact interface during mixing, resulting in heterogeneous aggregates. Here, we succeeded in preparing bulk PEC materials based on desalting-induced polyelectrolyte complexation via viscoelastic phase separation. With a high ionic strength aqueous medium, a homogeneous and concentrated solution containing oppositely charged polyelectrolytes is prepared. Desalting of the counter-ions and co-ions of the solution through semi-permeable membranes induces viscoelastic phase separation of the solution to form a physical hydrogel with open pore structure. Regulating the charge ratio of the two oppositely charged polymers results in significant changes in the porous morphology and mechanical properties. The charge-balanced PEC hydrogels show unique properties including high toughness and self-recovery due to the reversible ionic associations. The porous yet tough properties of bulk PEC hydrogels makes them potential candidates for applications such as cell scaffolds.

Ginkgolic acid obtained as a sphingomyelin synthase inhibitor from a plant extract library inspired the concept of sphingolipid mimics. Ginkgolic acid-derived N-acyl anilines and ginkgolic acid 2-phosphate (GA2P) respectively mimic ceramide and sphingosine 1-phosphate (S1P) in structure and function. The GA2P-induced phosphorylation of ERK and internalization of S1P receptor 1 (S1P1) indicated potent agonist activity. Docking studies revealed that GA2P adopts a similar binding conformation to the bound ligand ML5, which is a strong antagonist of S1P1.

BackgroundDespite the high prevalence of genotype 1b hepatitis C virus (HCV) among patients, a cell culture system that permits entire viral life cycle of genotype 1b isolates is limited. To develop a cell-cultured hepatitis C virus (HCVcc) of genotype 1b, the proper combination of HCV genomic variants and host cells is essential. HCV genomes isolated from patients with distinctive symptoms may provide the variants required to establish an HCVcc of genotype 1b.ResultsWe first established subgenomic replicons in Huh7 cells using HCV cDNAs isolated from two patients: one with fulminant hepatitis after liver transplantation (TPF1) and another with acute hepatitis and moderate symptoms (sAH). Replicons established from TPF1 and sAH showed mutations in NS4B and in NS3 and NS5A, respectively. Using these replication machineries, we constructed HCV genomic RNAs for each isolate. Virus infectivity was evaluated by a focus-forming assay, which is dependent on the intracellular expression of core antigen, and production of virus particles was assessed by density-gradient centrifugation. Infectious virus was only observed in the culture medium of cells transfected with TFP1 HCV RNA. A chimeric genome with the structural segment (5′-untranslated region [UTR] through NS2) from sAH and the replication machinery (NS3 through 3′-UTR) from TPF1 exhibited greater infectivity than did TFP1, despite formation of deficient virus particles in sAH, suggesting that this genomic segment potentiates virus particle formation. To identify the responsible variants, infectious virus formation was assessed in a chimeric genome carrying parts of the sAH structural segment of the TPF1 genome. A variant in NS2 (M170T) was identified that enhanced infectious virus formation. HCVcc carrying an NS2 gene encoding the M170T substitution and adaptive mutations in NS4B (referred to as TPF1-M170T) infected naïve cured Huh7 cells in a CD81-dependent manner.ConclusionsWe established a novel HCVcc of genotype 1b in Huh7 cells by introducing an amino acid variant in NS2 and adaptive mutations in NS4B from HCV genomic RNA isolated from a patient with fulminant HCV after liver transplantation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0846-9) contains supplementary material, which is available to authorized users.

Hepatitis B surface antigen (HBsAg) is used as a clinical marker of hepatitis B virus (HBV) infection. However, conventional HBsAg assays have so far failed to accurately detect HBsAg in blood because of interference by patient-derived antibodies against HBsAg (HBsAb). We developed a novel, fully automated assay system that can detect total HBsAg in blood, including antigens complexed with HBsAb. The immunoassay inactivates HBsAb via a simple pretreatment step to dissociate the HBsAg molecule from HBsAg-HBsAb complexes and thereby estimate total HBsAg. Accordingly, the test has been termed the "immunoassay for total antigen including complex via pretreatment (iTACT)-HBsAg." The recovery rate of HBsAg in the presence of HBsAb was greater than 87% at a cutoff value set at 5.0mIU/mL on the basis of data from 545 healthy controls. Analyses using serial serum samples from 25 HBV carriers who became negative for HBsAg during follow-up showed that the iTACT-HBsAg could detect HBsAg over a period of years despite a loss in detection by conventional assays and was able to detect HBsAg in 39 (53%) of 73 samples with HBsAb. The new iTACT-HBsAg assay appears to detect total HBsAg with high sensitivity, even in the presence of HBsAb, and may useful in identifying subclinical or occult HBV carriers.

Background and Aims: The hepatitis C virus (HCV) genotype 1b is known to exhibit treatment resistance with respect to interferon (IFN) therapy. Substitution of amino acids 70 and 91 in the core region of the 1b genotype is a significant predictor of liver carcinogenesis and poor response to pegylated-IFN-α and ribavirin therapy. However, the molecular mechanism has not yet been clearly elucidated because of limitations of the HCV genotype 1b infectious model. Recently, the TPF1-M170T HCV genotype 1b cell culture system was established, in which the clone successfully replicates and infects Huh-7-derived Huh7-ALS32.50 cells. Therefore, the purpose of this study was to compare IFN resistance in various HCV clones using this system. Methods: HCV core amino acid substitutions R70Q and L91M were introduced to the TPF1-M170T clone and then transfected into Huh7-ALS32.50 cells. To evaluate the production of each virus, intracellular HCV core antigens were measured. Results were confirmed with Western blot analysis using anti-NS5A antibodies, and IFN sensitivity was subsequently measured. Results: Each clone was transfected successfully compared with JFH-1, with a significant difference in intracellular HCV core antigen (p < 0.05), an indicator of continuous HCV replication. Among all clones, L91M showed the highest increase in the HCV core antigen and HCV protein. There was no significant resistance against IFN treatment in core substitutions; however, IFN sensitivity was significantly different between the wildtype core and JFH-1 (p < 0.05). Conclusions: A novel genotype 1b HCV cell culture was constructed with core amino acid substitutions, which demonstrated IFN resistance of genotype 1b. This system will be useful for future analyses into the mechanisms of HCV genotype 1b treatment.

Most studies on hydrogel swelling instability have been focused on a constrained boundary condition. In this paper, we studied the mechanical instability of a piece of disc-shaped hydrogel during free swelling. The fast swelling of the gel induces two swelling mismatches; a surface-inner layer mismatch and an annulus-disc mismatch, which lead to the formation of a surface crease pattern and a saddle-like bulk bending, respectively. For the first time, a stripe-like surface crease that is at a right angle on the two surfaces of the gel was observed. This stripe pattern is related to the mechanical coupling of surface instability and bulk bending, which is justified by investigating the swelling-induced surface pattern on thin hydrogel sheets fixed onto a saddle-shaped substrate prior to swelling. A theoretical mechanism based on an energy model was developed to show an anisotropic stripe-like surface crease pattern on a saddle-shaped surface. These results might be helpful to develop novel strategies for controlling crease patterns on soft and wet materials by changing their three-dimensional shape.

Marine collagen has been attracting attention as a medical material in recent times due to the low risk of pathogen infection compared to animal collagen. Type I collagen extracted from the swim bladder of Bester sturgeon fish has excellent characteristics such as high denaturation temperature, high solubility, low viscosity and an extremely fast rate to form large bundle of fibers under certain conditions. These specific characteristics of swim bladder collagen (SBC) permit us to create stable, disk shaped hydrogels with concentric orientation of collagen fibers by the controlled diffusion of neutral buffer through collagen solution at room temperature. However, traditionally used animal collagens, e.g. calf skin collagen (CSC) and porcine skin collagen (PSC), could not form any stable and oriented structure by this method. The mechanism of the superstructure formation of SBC by a diffusion induced gelation process has been explored. The fast fibrillogenesis rate of SBC causes a quick squeezing out of the solvent from the gel phase to the sol phase during gelation, which builds an internal stress at the gel-sol interface. The tensile stress induces the collagen molecules of the gel phase to align along the gel-sol interface direction to give this concentric ring-shaped orientation pattern. On the other hand, the slow fibrillogenesis rate of animal collagens due to the high viscosity of the solution does not favor the ordered structure formation. The denaturation temperature of SBC increases significantly from 31 °C to 43 °C after gelation, whereas that of CSC and PSC were found to increase a little. Rheology experiment shows that the SBC gel has storage modulus larger than 15 kPa. The SBC hydrogels with thermal and mechanical stability have potential as bio-materials for tissue engineering applications.