An overview of the production of tissue extracellular matrix and decellularization process.

Thousands of patients need an organ transplant yearly, while only a tiny percentage have this chance to receive a tissue/organ transplant. Nowadays, decellularized animal tissue is one of the most widely used methods to produce engineered scaffolds for transplantation. Decellularization is defined as physically or chemically removing cellular components from tissues while retaining structural and functional extracellular matrix (ECM) components and creating an ECM-derived scaffold. Then, decellularized scaffolds could be reseeded with different cells to fabricate an autologous graft. Effective decellularization methods preserve ECM structure and bioactivity through the application of the agents and techniques used throughout the process. The most valuable agents for the decellularization process depend on biological properties, cellular density, and the thickness of the desired tissue. ECM-derived scaffolds from various mammalian tissues have been recently used in research and preclinical applications in tissue engineering. Many studies have shown that decellularized ECM-derived scaffolds could be obtained from tissues and organs such as the liver, cartilage, bone, kidney, lung, and skin. This review addresses the significance of ECM in organisms and various decellularization agents utilized to prepare the ECM. Also, we describe the current knowledge of the decellularization of different tissues and their applications.

Designing a bioadjuvant candidate vaccine targeting infectious bursal disease virus (IBDV) using viral VP2 fusion and chicken IL-2 antigenic epitope: A bioinformatics approach.

Infectious Bursal Disease (IBD) is a common and contagious viral infection that significantly affects the poultry industry. This severely suppresses the immune system in chickens, thereby threating their health and well-being. Vaccination is the most effective strategy for preventing and controlling this infectious agent. The development of VP2-based DNA vaccines combined with biological adjuvants has recently received considerable attention due to their effectiveness in eliciting both humoral and cellular immune responses. In this study, we applied bioinformatics tools to design a fused bioadjuvant candidate vaccine from the full-length sequence of the VP2 protein of IBDV isolated in Iran using the antigenic epitope of chicken IL-2 (chiIL-2). Furthermore, to improve the antigenic epitope presentation and to maintain the three-dimensional structure of the chimeric gene construct, the P2A linker (L) was used to fuse the two fragments. Our in-silico analysis for the design of a candidate vaccine indicates that a continuous sequence of amino acid residues ranging from 105 to 129 in chiIL-2 is proposed as a B cell epitope by epitope prediction servers. The final 3D structure of the VP2-L-chiIL-2105-129 was subjected to physicochemical property determination, molecular dynamic simulation, and antigenic site determination. The results of these analyses led to the development of a stable candidate vaccine that is non-allergenic and has the potential for antigenic surface display potential and adjuvant activity. Finally, it is necessary to investigate the immune response induced by our proposed vaccine in avian hosts. Notably, increasing the immunogenicity of DNA vaccines can be achieved by combining antigenic proteins with molecular adjuvants using the principle of rational vaccine design.

The in vitro analysis of migration and polarity of blastema cells in the extracellular matrix derived from bovine mesenteric in the presence of fibronectin.

Cell migration is an essential process in embryonic development, wound healing, and pathological conditions. Our knowledge of cell migration is often based on the two dimentional evaluation of cell movement, which usually differs from what occurred in vivo. In this study, we investigated cellular migration from blastema tissue toward bovine decellularized mesentery tissue. In this regard, fibronectin (FN) was assessed to confirm cell migration. Therefore, we established a cell migration model using blastema cells migration toward the extracellular matrix derived from bovine mesenteric tissue. A physiochemical decellularization method was utilized based on freeze-thaw cycles and agitation in sodium dodecyl sulfate and Triton X-100 to remove cells from the extracellular matrix (ECM) of bovine mesenteric tissue. These types of matrices were assembled by the rings of blastema tissues originated from the of New Zealand rabbits pinna and cultured in a medium containing FN in different days in vitro, and then they are histologically evaluated, and the expression of the Tenascin C gene is analyzed. By means of tissue staining and after confirmation of the cell removal from mesenteric tissue, polarity, and migration of blastema cells was observed in the interaction site with this matrix. Also, the expression of the Tenascin C gene was assessed on days 15 and 21 following the cell culture process. The results showed that the three dimentional model of cellular migration of blastema cells along with the ECM could be a suitable model for investigating cell behaviors, such as polarity and cell migration in vitro.

An overview of progress from empirical to rational design in modern vaccine development, with an emphasis on computational tools and immunoinformatics approaches.

Vaccination remains the most effective strategy for preventing and controlling infectious diseases. Numerous conventional vaccines, especially live attenuated, inactivated (killed) microorganisms and subunit vaccines, lead to an effective induction of protective immune responses, mainly antibody-mediated responses against pathogens. However, it has become known that a wide range of highly dangerous pathogens are uncontrollable via conventional vaccination strategies. Recent advances in molecular biology, immunology, genetics, biochemistry, and bioinformatics have provided new prospects for vaccine development. As a result of these advances, several new strategies for vaccine design, development, and production have appeared. These strategies show advantages over conventional vaccines. In this review, we discuss some of the major novel approaches, including recombinant protein vaccines, live recombinant viral and bacterial vectors, DNA and RNA vaccines, reverse vaccinology and reverse genetics approaches. Moreover, we have described the recent progresses on computational tools and immunoinformatics approaches for identifying, designing, and developing new candidate vaccines.

Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype.

One of the major bacterial infectious diseases in the poultry industry is avian pathogenic Escherichia coli (APEC), which causes colibacillosis in chickens. To develop a novel nucleic acid-free bacterial ghost (BG) vaccine against the O78:K80 serotype of APEC, in this study we constructed a plasmid that harbored E-lysis and S nuclease (SNUC). Following the expression, the O78:K80 bacteria lost all of their cytoplasmic content and nucleic acids by enzymatic digestion. The functionality of these two proteins in the production procedure of bacterial ghosts was confirmed by monitoring the number of colonies, scanning electron microscopy imaging, gel electrophoresis of genomic DNA, and qPCR on the plasmid content of bacterial ghosts. The protective efficacy of the ghost vaccine generated from O78:K80 serotype of APEC was tested in chickens by injection and inhalation routes and compared with that in chickens that received the injection of a killed vaccine. The O78:K80 BG vaccine candidate, used as injection and inhalation, in comparison with the killed vaccine, triggered higher proinflammatory cytokine expression including IL-6, IL-1ß, and TNFSF15; a higher level of antibody-dependent humoral (IgY and IgA) and cellular immune responses (IFNγ and lymphocyte proliferation); and lower lesion scores. According to the results of this study, we suggest that the bacterial ghost technology has the potential to be applied for the development of novel vaccines against avian colibacillosis. This technology provides an effective and reliable approach to make multivalent vaccines for more prevalent APEC strains involved in the establishment of this infectious disease in the poultry industry.

Reverse Genetics Assembly of Newcastle Disease Virus Genome Template Using Asis-Sal-Pac BioBrick Strategy.

BACKGROUND: The BioBrick construction as an approach in synthetic biology provides the ability to assemble various gene fragments. To date, different BioBrick strategies have been exploited for assembly and cloning of a variety of gene fragments. We present a new BioBrick strategy, here referred as Asis-Sal-Pac BioBrick, which we used for the assembly of NDV as a candidate for single-stranded non-segmented, negative-sense RNA genome viruses. RESULTS: In the present study, we isolated three NDVs from clinical samples which were classified into the VIId genotype based on their pathogenicity and phylogenetic analyses. Then, SalI, AsisI, and PacI enzymes were used to design and develop a novel BioBrick strategy, which enabled us to assemble the NDV genome, adopting the "rule of six". In this method, in each assembly step, the restriction sites in the newly formed destination plasmid are reproduced, which will be used for the next insertion. In this study using two overlapping PCRs, the cleavage site of the F gene was also modified from 112RRQKRF117to 112GRQGRL117 in order to generate the attenuated recombinant NDV. Finally, in order to construct the recombinant NDV viruses, the plasmids harboring the assembled full-length genome of the NDV and the helper plasmids were co-transfected into T7-BHK cells. The rescue of the recombinant NDVwas confirmed by RT-PCR and HA tests. CONCLUSIONS: These findings suggest that the combination of reverse genetic technology and BioBrick assembly have the potential to be applied for the development of novel vaccine candidates. This promising strategy provides an effective and reliable approach to make genotype-matched vaccines against specific NDV strains or any other virus.

Efficient Media for High Lipase Production: One Variable at a Time Approach.

BACKGROUND: Lipase enzymes have applications in a wide range of industries. A crucial determining factor of industrial prices of these enzymes is the culture media composition that is constantly under review by researchers. In this work, for maximum lipase production by Bacillus sp. ZR-5, culture media compositions were optimized using ″one variable at a time″ strategy. METHODS: For this purpose, the culture medium parameters such as low and high cost carbon and nitrogen sources, substrates and incubation times were evaluated. RESULTS: Maximum lipase activity was achieved after 24 hr of incubation with 1.5% of glucose syrup (1600±69.1 u/mg), 1% of fish powder (1238±36.7 u/mg) and olive oil (1407±2.1 u/mg) as low cost carbon and nitrogen sources and substrate, respectively. CONCLUSION: Our results show a significant increase in lipase activity with usage of low cost sources; this could help in reducing the media prices for industrial application of lipase enzyme.

Inactivation of aprE Gene in Bacillus subtilis 168 by Homologus Recombination.

BACKGROUND: One of the most important producers of high quality industrial enzymes is the Gram-positive bacterium, Bacillus subtilis (B. Subtilis). One major limitation that hinders the wide application of B. subtilis is the secretion of high levels of extracellular proteases which degrade the secreted foreign proteins. In this study, homologus recombination technique was used to knock out its protease gene, aprE. METHODS: The internal segment of the pro-sequence of aprE gene of B. subtilis 168 with a length of 80 bps and its complementary sequence were synthesized and ligated into pUB110 at EcoR1 and XbaI restriction sites. Competent cells of B. subtilis 168 were prepared and transformed by electroporation using Bio Rad gene pulser as explained in the methods section. Transformants carrying the recombinant plasmid were selected for resistance to neomycin. The success of homologous recombination was checked by PCR amplification of the neomycin gene which was part of the vector and did not exist in the genome of B. subtilis 168. The protease activity was measured using the Protease Fluorescent Detection Kit based on the proteolytic hydrolysis of fluorescein isothiocyanate (FITC)-labeled casein-substrate. RESULTS: The results demonstrated that aprE gene would not be able to produce further active subtilisin E. The reduction of protease activity also confirmed the efficacy of the induced mutation in this gene. CONCLUSION: It will therefore be a major challenge for future research to identify and modulate quality control systems of B. subtilis which limit the production of high quality protease- sensitive products such as lipase.