Development of a Novel Subunit Vaccine Targeting Fusobacterium nucleatum FomA Porin Based on In Silico Analysis

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.

Ideal Freezing Curve Can Avoid the Damage by Latent Heat of Fusion During Freezing / 대한흉부외과학회지

BACKGROUND: Liquid nitrogen freezing techniques have already met with widespread success in biology and medicine as a means of long-term storage for cells and tissues. The use of cryoprotectants such as glycerol and dimethylsulphoxide to prevent ice crystal formation, with carefully controlled rates of freezing and thawing, allows both structure and viability to be retained almost indefinitely. Cryopreservation of various tissues has various controlled rates of freezing. MATERIAL AND METHOD: To find the optimal freezing curve and the chamber temperature, we approached the thermodynamic calculation of tissues in two ways. One is the direct calculation method. We should know the thermophysical characteristics of all components, latent heat of fusion, area, density and volume, etc. This kind of calculation is so sophisticated and some variables may not be determined. The other is the indirect calculation method. We performed the tissue freezing with already used freezing curve and we observed the actualfreezing curve of that tissue. And we modified the freezing curve with several steps of calculation, polynomial regression analysis, time constant calculation, thermal response calculation and inverse calculation of chamber temperature. RESULT: We applied that freezing program on mesenchymal stem cell, chondrocyte, and osteoblast. The tissue temperature decreased according to the ideal freezing curve without temperature rising. We did not find any differences in survival. The reason is postulated to be that freezing material is too small and contains cellular components. We expect the significant difference in cellular viability if the freezing curve is applied on a large scale of tissues. CONCLUSION: This program would be helpful in finding the chamber temperature for the ideal freezing curve easily.

Safety of Aprotinin Under Hypothermic Circulatory Arrest / 대한흉부외과학회지

It was reported that use of aprotinin in elderly patients undergoing hypothermic circulatory arrest was associated with an increased risk of renal dysfunction, and myocardial infarction as a result of intravascular coagulation. We reviewed 20 patients who received high-dose aprotinin under deep hypothermic circulatory arrest with(NP group, n=11) or without selective cerebral perfusion(SP group, n=9). The activated clotting time was exceeded 750 seconds in all but 1 patient. After opening aortic arch, retrograde low flow perfusion was maintained through femoral artery to prevent air embolization to the visceral arteries. Four patients among 20 died during hospitalization due to bleeding, coronary artery dissection, pulmonary hemorrhage and multiple cerebral infarction. Postoperatively, cerebrovascular accidents occurred in two patients; one with preoperative carotid artery dissection and the other with unknown multiple cerebral infarction. In conclusion, use of aprotinin in young patients undergoing hypothermic circulatory arrest did not increase the risk of renal dysfunction or intravascular coagulation if ACT during circulatory arrest is maintained to exceed 750 seconds with low-flow perfusion.