Journal of Applied Biotechnology Reports
Volume:6 Issue: 1, Winter 2019

Regenerative medicine (RM) is a developing multidisciplinary science that uses different principles and methods of other fields or sciences for tissue regeneration, repair or replacement. Gene therapy refers to transferring genes or gene expression regulator factors for the desired purposes. In some cases, gene therapy plays an important role in regenerative medicine by modulating stem cells from different sources. Genetic heterogeneity of individuals can affects the results of gene therapy or other therapies in RM. This is why personal genomics should be considered in RM and is called personalized regenerative medicine (PRM). The purpose of PRM is to employ strategies and methods tailored to the individual’s genetics in order to efficiently reconstruct or substitute various parts of the body. In this study, the strategies and recent advances in bone regeneration such as gene therapy, epigenetic-based therapies, RNA-based therapy and CRISPR/Cas9 system with an attitude to personalized medicine are introduced.

Recombinant proteins have become one of the basic human needs today for pharmaceutical, industrial and research purposes. The conventional methods of producing recombinant proteins, including bacterial systems, yeast, and human cells, have led to rising prices and biosafety problems for these products. Research on commercial production of recombinant proteins prompted a new method based on plant RNA viruses and plant hosts, which is known as Transient Expression System. Accordingly, a viral vector based on the plant RNA virus genome was designed in which the target gene is placed underneath a viral subgenomic promoter. In these vectors, despite the genetic manipulation, the ability to infect the entire plant that was present in the virus was preserved and, several days after plant infestation, it became bioreactor for the production of recombinant protein. The advances made in this field led to the creation of hybrid vectors based on plant viruses and Agrobacterium T-DNA (Transfer DNA). The amount of recombinant protein produced by these vectors was up to 5 grams per kilogram of fresh weight, which is considered a reliable record. The abundant benefits of this method have attracted the attention of researchers in this way, including the rapid and high production rate, reduced production costs and bio-safety of manufactured products. Several recombinant drugs are currently produced by the transient expression system, either delivered to the consumer or by clinical trials. This review presents how to discover, creation and development of viral vectors in transient expression systems in order to produce recombinant proteins.

Honey bees are used intensively to boost the agricultural and economic sectors worldwide. Many viruses attack honey bees and cause severe problems to the bee colonies, and constitute a real challenge for beekeeping development. Hence, understanding the genetic characteristics of bee viruses is necessary to highlight the phylogenetic relationships between them, and to find out similarity aspects based on sequences. Some public resources and free genetic analysis programs were utilized to perform this study. The complete sequences for some viruses were downloaded and analyzed using various programs and methods. Some viruses shared the same base composition pattern in regards to percentage of A, T, C, and G. The phylogenetic relationships among the investigated viruses were presented and discussed. The phylogenetic trees constructed using three bioinformatics programs based on different methods emphasized the relationship between Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV), and between deformed wing virus (DWV) and Kakugo virus (KV). These genetic relationships were also confirmed using enzymatic digestion to the sequences, gene cluster families, and open reading frames (ORFs). This study has presented new trends to analyze genetic similarities between organisms utilizing sequences. Different results for the phylogenetic relationships could be obtained when performing the analysis using various methods without impacting the relationships between closely related organisms. This study encourages the performance of additional studies to figure out functional components of these viruses.

Organophosphorus hydrolase(OPH) is an enzyme that can degrade organophosphorus compounds in pesticides. High expression levels of OPH in Escherichia coli lead to form inclusion body in cytoplasmic space which is an inactive form of protein and needs a solubilizing and refolding process. The aim of this study was to compare different methods for solubilization and refolding of recombinant OPH expressed in E. coli. OPH was expressed in E. coli and purified by the Ni-NTA column. The refolding efficiency of this protein was assessed by 4 strategies: dialysis, rapid dilution, on column and combination of rapid dilution and dialysis. In each case, the refolding efficiency was evaluated by SDS-PAGE analysis and enzyme activity assay and was compared to find the best procedure. The refolding efficiency of these 4 strategies was estimated at about 12%, 10%, 14% and 50% for on column, rapid dilution, dialysis and combination of rapid dilution and dialysis, respectively. Results showed that during the refolding process, most proteins did not reach the correct structure and aggregated again while the combination of 2 methods, rapid dilution and dialysis provided an appropriate procedure to refold. The combination of rapid dilution and dialysis is an efficient method for refolding OPH. The efficacy of this method for refolding other recombinant proteins can be further investigated.

White rot, caused by Sclerotinia sclerotiorum, has recently become a serious threat to tuber mustard cultivation in Hangzhou, China. The objective of this study was to evaluate the inhibitory effect of acetyl salicylic acid (ASA) and three different chitosans (A, B and C) against mustard white rot. The degree of N-deacetylation and the molecular weight of chitosans A, B and C were 85%-1129 kDa, 95%-521 kDa and 75%-607 kDa, respectively. The inhibitory effect of chitosans with different concentrations against the mycelia growth and sclerotia formation of 3 isolates of the pathogen was determined in vitro. In addition, the efficacy of chitosans and ASA against mustard white rot was assessed during in vivo tests. After protein extraction, effects of chitosans and ASA on resistance related enzymes including chitinase, β-1,3-glucanase, phenylalanine ammonia lyase, polyphenol oxidase (PPO) and peroxidase (POD) were evaluated. The chitosans reduced the mycelia growth and sclerotia formation of the pathogen. The chitosans showed significant antifungal effect against the disease in vivo. The chitosans and ASA markedly reduced the severity of the disease over time. Moreover, the chitosans and ASA markedly enhanced the level of most of the resistant related enzymes after 3 and 6 days. The chitosan B was found to have the best effect against tested pathogen isolates. The chitosan with the lowest molecular weight was found to be more effective against the disease. In addition, chitosans and ASA were able to significantly increase resistance-related enzymes over time indicating that they can be considered as resistant inducers against mustard white rot.

Bacillus thuringiensis var kurstaki is one of the best known biological insecticide which is used extensively in the world. This biopesticide has been broadly used against important insect pests and vectors of animals and humans. However, the controversial data was published to date considering toxicological studies on non-target species are urgent to determine probable adverse effects and risk assessment of this biopesticide. In this research, histopathological changes, hematological and some biochemical factors were evaluated following the single dose oral administration of B. thuringiensis in rats. Twelve Wistar rats were randomly divided into 2 groups including experimental and control groups. Animals were treated orally by gavage to single dose of sub-lethal dose (5000 mg/kg) of B. thuringiensis suspension. Finally, hematological factors (WBC, RBC, Hb, HCT, MCV, MCH, MCHC and PLT), some biochemical parameters (ALT, AST, ALP, BUN and Cr) and histopathological observations were evaluated. The results demonstrated that oral administration of high dose of B. thuringiensis biopesticide induced some pathological complications including congestion and inflammation in vital organs of rats such as liver, heart, lung and kidney. It also caused hematological abnormalities and biochemical alterations in the experimental animals group. According to the findings, it can be concluded that high dose of B. thuringiensis biopesticide can induce toxicity in rats. Therefore, further investigations including subacute and chronic are recommended.

Arsenic toxicity has posed troublesome health concerns in the world and many of the toxic effects of arsenic are related to its effect on oxidative stress. The aim of the present study is to evaluate histopathological and antioxidant enzymes changes in oxidative stress status induced by sodium arsenite in rats. All experiments were carried out in male Wistar rats. Animal were divided into 2 groups of eight animals in each: Rats consumed distilled water (control group). Group 2: Rats consumed a solution of sodium arsenite (100 ppm) daily (arsenic group). At the end of day 28 arsenic exposure, the enzyme level in rat liver and kidney tissues was measured using the assay kits. The remaining liver, kidney, and heart tissue were fixed in 10% neutral-buffered formalin and used for histological observation. The results showed a significant decrease in values of glutathione peroxidase (GPx), superoxide dismutase (SOD), total antioxidant capacity (TAC) and catalase (CAT) in serum and tissue in kidney and liver rat (P < 0.05). But, malondialdehyde (MDA) levels were increased significantly (P < 0.05). Arsenic caused severe degenerative changes in tubular cells and acute tubular necrosis, hepatocyte cell degeneration, severe hemorrhage, and infiltration and formation of Kupffer cells nodules, fragmentation, and degeneration of muscle fibers with pyknotic nuclei in heart tissue. The finding of the present study revealed that the administration of Sodium arsenite caused significant oxidative stress, decreased antioxidant enzymes activity and severe tissue damage.