Modares Journal of Biotechnology

Abstract Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease and the prevalence of it has increased worldwide. PD causes severe damage to loss of midbrain dopaminergic neurons (DN) at substantia nigra which involves in the movement control. The synaptic terminals of DNs are destroyed in the posterior putamen. PD is also characterized by accumulation of misfolded and amyloid α-synuclein into proteinous inclusions named Lewy bodies. So far there is no effective treatment for PD and approved medications for PD can only slow down clinical progression, control motor and non-motor symptoms. Currently, the approved medications just induce the release of dopamine and prevent the release of acetylcholine from nerve terminals of caudate cholinergic interneurons and it is necessary to provide more effective treatment methods in the early stages of the disease. Significant progress has been achieved in development of drugs that can reduce neuronal cell death and lead up to neuroprotection, however targeting delivery of drugs to improve the pharmaceutical effects of drugs is remained as a challenge. Therefore, there is a pressing demand to find practical strategies for delivering these pharmaceuticals in vivo through the BBB without disrupting the brain's functions. In this regard, Focused ultrasound (FUS) technology eliminates the need for brain surgery and temporarily opens the blood-brain barrier to allow drugs to pass through. In this review, the application of FUS as a new drug delivery application in the PD models and the potential clinical application of neuroprotective agents are provided briefly.

Abstract The process of diphtheria toxoid production was designed by using SuperPro Designer and the effect of the applied changes in process on the yield and costs of the manufacturing was investigated. First, giving the information of the real process of the toxoid production, a bioreactor with improved operational conditions and a disc stack centrifuge instead of the filter press, which is applied for the bacterial debris separation, were utilized. Such alterations followed the addition of a pump between the bioreactor and centrifuge. The results indicated that improvement of the bioreactor operational conditions can lead to the 25% increase in the toxin production, i.e., the increase of toxoid production from 7,000,000 doses to 8,750,000 doses. The toxin waste through filter press (14%) may be remarkably reduced by using the centrifuge, which in turn resulted in the 44% enhancement in the toxoid production. Such alterations can result in the 16% reduction in the separation operation time, 29% reduction in water consumption and 32% increase in the energy consumption. Overall, the simulation results showed that the costs of the new equipment suggested to be used in the improved process can be recoverable through running two batches.

Abstract In recent years, biocatalysts have widespread application in industry because they can do chemical reactions with the lowest energy and highest efficiency. Bacterial enzymes are more useful in this field due to simple cloning and expression process in the manipulated host. By considering specific role of endoglucanase enzymes in cellulose hydrolyzing reactions, these types of enzymes are more applicable in related industries. The produced glucose through enzymatic hydrolysis could be used in different industries such as biofuel and ethanol production and in the food industry as sweetener. Therefore, cloning and production of Endoglucanase in manipulated hosts has been developed in recent years. This study was performed to isolate, screen and identify native endoglucanase -producing strains from soil around the roots of the maple tree. Isolated strains were identified using 16S rRNA gene sequencing. After identifying of the bacteria (Enterobacter hormaechei), Endoglucanase enzyme gene was amplified using degenerate primers at first and then by specific primers with restriction enzymes sequences. DNA fragment and plasmid vector were treated by specific restriction enzymes and then ligated to each other. Then recombinant plasmid transferred to the E. coli BL-21 as expression host and kinetic properties of recombinant enzyme were evaluated. Expression of the target protein was done by stimulating the Lac operon by using 1 mM of IPTG and the kinetic features of the recombinant enzyme such as Vmax and Km evaluated as 45 µmol/min and 1.4 mg/ml respectively. The optimum conditions for enzyme activity tend to be 37°C at a pH of 7.

Abstract Transthyretin (TTR) is a highly conserved 55 kDa homotetrameric protein that exists in several vertebrate species including humans, bacteria, nematodes, and plants. Previous studies have shown a direct interaction between TTR and amyloid beta (Aβ) (the causative agent of Alzheimer's disease), which leads to the inhibition of Aβ aggregation, fibrillar destruction, or both. In recent years, evidence has shown that the oligomeric species of Aβ formed by the aggregation process are more toxic than mature fibrils. Studies have shown that such an oligomeric mediator is modulated by interaction with TTR. However, the exact mechanism of binding of Aβ to TTR has not yet been determined. In this study, after the purification of human transthyretin protein, the inhibitory effects of TTR on the formation of Aβ were shown in different ways, and finally, the role of hydrophobicity interactions in the chaperone activity of TTR was investigated with the help of protein surface hydrophobicity (PSH) measurement studies. The Scatchard diagram for quantitative measurement of PSH indicates an increase in the hydrophobicity of TTR after binding to oligomeric forms of Aβ. The results presented in this research provide insight into the nature and interactions involved in the initial stages of fibril formation in Aβ and its interaction with TTR. The results showed that hydrophobic interactions probably play a role in the binding between TTR and Aβ. Considering the similarity of amyloid formation systems, the described findings of this study can provide a deeper understanding of the pathology of amyloid diseases.

Abstract Over the past few years, due to the shortage of forest resources as well as the increasing consumption of paper and paperboard, many pulp and paper manufacturers have developed the use of different sources of recycled (secondary) fibers. Therefore, recycling of paper is an effective and eco-friendly way to preserve forest resources, which eventually save the natural diversity and energy. The use of biotechnology in various sectors of the cellulosic products industries such as bio-pulping, bio-bleaching, bio-deinking, bio-wastewater treatment, etc. has been considered and good achievements have been obtained in this field. One of the most important applications of biotechnology in the cellulosic industries is the use of enzymes in the processing of recycled fibers. The use of enzymatic technologies, as environmental friendly process, has led to changes in industrial processes as much as possible and indicated great potential in solving many problems of recycled fibers, especially problems related to waste paper deinking, pulp drainage rate, fiber hornification, refining and stickies materials. In general, deinking with enzymes under acidic or neutral conditions declines the chemicals usage and reduces the yellowing of recycled paper under conventional alkaline deinking conditions. Today, the use of cellulosic enzymes (cellulase and hemicellulase) and oxidative enzymes (such as laccase) as well as amylase and pectinase have shown acceptable results for deinking different types of waste paper and most experiments in semi-industrial as well as industrial units have shown that deinking with them can reduce the cost of chemicals, increase the separation of ink particles

Abstract The volatility of essential oils and their instability against environmental factors limit their use. However, encapsulating these compounds in polymeric nanoparticles can significantly increase their half-life and make their use possible for longer periods. Chitosan, a biodegradable polymer with controlled release and low toxicity, is one of the polymers utilized in the encapsulation of essential oils. As a result, the present study was carried out with the aim of nanoencapsulation of carvacrol and thymol in chitosan using ionic gelation method and the concentration of chitosan (0.1-0.3%), TPP concentration (0.2-0.1%) and concentration of essential oil (0.1-0.2%) as three variables. The mean particle size of chitosan nanoparticles was optimized using response surface methodology and central composite design. The size and polydispersity index (PDI) were determined by DLS, essential oil loading confirmation was evaluated by FTIR, and the spectrophotometric method was used to measure the encapsulation efficiency. Then, the results of the optimization of nanoparticle synthesis were investigated. The optimum conditions for the synthesis of chitosan-thymol nanoparticles and achieving a size of 101 nm and encapsulation efficiency of 72%, a concentration of 0.11% chitosan, 0.19% TPP and 0.14% thymol were determined. In the case of chitosan-carvacrol nanoparticles, a concentration of 0.13% chitosan, 0.19% TPP and 0.15% carvacrol resulted in the formation of nanoparticles with a size of 95 nm and an encapsulation efficiency of 65%. In general, the results demonstrated the ability of the response surface methodology to predict the particle size and PDI of chitosan nano-formulations containing carvacrol and thymol.

Abstract Angiogenesis is involved in many physiological and pathological processes, including tumor growth, and VEGF is considered as the most important factor in this process. Nowadays, the production of single-domain antibodies (VHH) with the characteristic of inhibiting growth factors in cancer tumors is one of the new strategies for cancer treatment. In the previous research, it was found that camel VHHs isolated from phage display against VEGF play an essential role in inhibiting it. Here, the VHH that had the highest affinity for the VEGF was selected. Ice nucleation protein (INP) was used as anchoring motif for surface expression of E. coli, owing to the efficiency of its N terminal domain, this system is used to express the VHH. Accordingly, a construct harboring the first 537 nucleotide of InaK gene and nucleotide fragments of TEV protease recognition site and VEvhh10 was designed to express this protein in the surface display of E. coli cells. The results showed that the INP anchor is a suitable candidate for promoting the surface expression of VEvhh10 in E. coli. After expression of VEvhh10, isolation and purification were performed using centrifugation and washing, and its binding to VEGF was investigated. The results showed that VEvhh10 successful bind to VEGF and it can be used for therapeutic applications and clinical diagnosis of patients in the future.

Abstract Chondroitinase ABCI is a bacterial lyase that degrades glycosaminoglycans and promotes axonal growth and functional improvement. However the stability and maintenance of this enzyme is very limited. One of the strategies to overcome this limitation is to immobilize the enzyme. In this research, chondroitinase ABCI (cABCI) from Proteus Vulgaris was immobilized on hydroxyapatite nanoparticles. Hydroxyapatite is a non-toxic ceramic biomaterial that has a high surface area, which is beneficial for loading a large amount of enzyme. Therefore, to increase the stability of chondroitinase ABCI, immobilization on hydroxyapatite nanoparticles for 4 hours through physical adsorption in phosphate buffer pH 5, 6.8, and 8 at 4^◦C was carried out. Enzyme immobilization on hydroxyapatite nanoparticles was then confirmed by field emission gun-scanning electron microscopy and UV-spectroscopy, before and after immobilization. Then, in order to obtain the optimal pH and temperature, the activity of the nanosystem was investigated at three pH and temperatures (4°C, 25°C, and 37°C). Results revealed higher activity at pH 5 and temperature 4 ◦C than the other pH and temperatures for the nanosystem. Based on the obtained results, which show the stability of the nanosystem at all three temperatures compared to the free enzyme, this nanosystem could be a potential candidate for clinical applications in future.