Iranian Journal of Biotechnology
Volume:18 Issue: 3, Summer 2020

As a therapeutic enzyme, urate oxidase is utilized in the reduction of uric acid in various conditions such asgout or tumor syndrome lysis. However, even bearing kinetical advantage over other counterparts, it suffers from structuralinstability most likely due to its subcellular and fungal origin.

In this research, by using rational design and introduction of de novo disulfide bridge in urate oxidase structure,we designed and created a thermostable urate oxidase for the first time.

Utilizing site-directed mutagenesis and only with one point mutation we constructed two separate mutants: Ala6Cys and Ser282Cys which covalently linked subunits of enzyme each other. Single mutation to cysteine created three inter-chain disulfide bridges and one hydrogen bond in Ala6Cys and two disulfide bridges in Ser282Cys.

Both mutants showed 10 °C increase in optimum activity compared to wild-type enzyme while the Km values for both increased by 50% and their specific activity compromised. The thermal stability of Ser282Cys increased remarkably by comparing Ala6Cys and wild-type enzymes. Estimation of half life for wild-type enzyme demonstrated 38.5 min, while for Ala6Cys and Ser282Cys were 138 and 115 min, respectively. Interestingly, the optimal pH of both mutants was broaden from 7 to 10, which could make them candidates for industrial applications.

It seemed that introducing disulfide bridges resulted in local and overall rigidity by bringing two adjacent sites of enzyme together and decreasing the conformational entropy of unfolding state is responsible for the enhancement of thermostability.

Mitochondrion is the main indicator of oocyte quality and one of the components of oocyte, which is sensitive to oxidative damage during the maturation process. Mitoquinone mesylate (MitoQ) is a strong antioxidant targeting mitochondria as well as anti-apoptotic agent. However, the effect of MitoQ on the quality of oocytes during in vitro maturation (IVM) is still unknown.

This study investigated the possible effects of MitoQ on maturation and developmental competency in mice oocytes.

The oocytes were collected at germinal vesicle stage from 6-8-week old female NMRI mice and then cultured in TCM-199 medium supplemented with 0, 0.01, 0.02 and 0.04 μM MitoQ. The sham group was treated with DMSO (0.01% v.v). Then intracellular Glutathione (GSH), reactive oxygen species (ROS) levels, mitochondria membrane potential (ΔΨm), as well as in vitro fertilization (IVF) rate in the 18-20 h matured oocytes and metaphase II (MII) oocytes (in vivo-control), were assessed.

The results showed that between three dose of MitoQ, the 0.02 μM significantly increased nuclear maturation rate, GSH level, fertilization rate and blastulation (92.6, 231.7, 90.19 and 81.66%, respectively) than the in vitro-control (71.14,152, 78.84 and 73.50%, respectively) and more comparable to that of the in vivo matured oocytes (100, 243.5, 92.10 and 83%, respectively). Also, the mitochondria membrane potential in the 0.02 μM MitoQ was significantly higher compared with those in the other groups (4.4). However, the intracellular ROS level in 0.02 μM MitoQ was significantly decreased(38.72%) compared to in vitro-control (82.2%) and was similar to the in vivo-control (33.5%).

The results indicated that supplementation of IVM medium with MitoQ (specially 0.02 μM) enhance maturationand fertilization rate. In conclusion, MitoQ might be considered as a novel component that could be added to IVM media.

Staphylococcus aureus (SA) is known as an important human pathogen, which is responsible for many cases of both hospital and community-acquired infections all over the world. Studying on drug resistance is regarded as an important prevention strategy regarding these types of infections.

The current study is aimed to assess the association between the single-nucleotide polymorphism (SNP) and resistance to antibiotics in the methicillin-resistant Staphylococcus aureus (MRSA) strains as well as the molecular typing of isolates, collected from the clinical samples.

We used the disc-diffusion method to test the isolates antibiotic resistance. In addition, the genotypes of staphylococcal cassette chromosome mec (SCCmec) in the Methicillin-resistant Staphylococcus aureus isolates were determined by multiplex -polymerase chain reaction (PCR). SNP was identified in the mecA gene using sequencing and amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) method.

The highest resistance was shown against oxacillin, and erythromycin and cephalexin. The most sensitive antibiotic was vancomycin (97%) and resistance to at least three antibiotic classes were identified in all isolates. Eighty six percent of isolates were positive for mecA gene and more than 50% of which were healthcare-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA). Moreover, SCCmec type 3, 1were the predominant strains of the identified MRSA. Also, 23 isolates (23%) were non-typable. By using the ARMS-PCR method, it was found that 10% of the clinical specimens had SNP in the mecA gene.

According to the Chi-square test (χ2), it reveals that the association between SNP in the mecA gene and oxacillin, cefoxitin, and erythromycin resistance was confirmed among clinical MRSA. Furthermore, there is a 95%probability of association between SNP and resistance to more than three antibiotics in MRSA strains.

Recently, many researchers from different fields of science have been used networks to analyze complex relational big data. The identification of which nodes are more important than the others, known as centrality analysis, is a key issue in biological network analysis. Although, several centralities have been introduced degree, closeness, andbetweenness centralities are the most popular. These centralities are based on the individual position of each node and the cooperation and synergies between nodes have been ignored.

Since in many cases, the network function is a consequence of cooperation and interaction between nodes, classical centralities were extended to a group of nodes instead of only individual nodes using cooperative game theory concepts. In this study, we analyze the protein interaction network inferred in rabies disease and rank gene products basedon group centrality measurements to identify the novel gene candidates.

For this purpose, we used a game-theoretic approach at three scenarios, where the power of a coalition of genes assessed using different criteria including the neighbors of genes in the network, and predefined importance of the genes in its neighborhood. The Shapley value of such a game was considered as a new centrality. In this study, we analyze the network of gene products implicates rabies. The network has 1059 nodes and 8844 edges and centrality analysis was performed using CINNA package in R software.

Based on three scenarios, we selected genes among the highest Shapley value that had low ranking from classical centralities. The enrichment analysis among the selected genes in scenario 1 indicates important pathways in rabies pathogenesis. Pair-wise correlation analysis reveals that changing the weights of nodes at different scenarios can significantly affect the results of ranking genes in the network.

A prior knowledge about the disease and the topology of the network, enable us to design an appropriate game and consequently infer some biological important nodes (genes) in the network. Obviously, a single centrality cannot capture all significant features embedded in the network.

Phylogenetics is a branch of bioinformatics that studies and models the evolutionary relationships between currently living species. A phylogenetic tree is the simplest possible model in which leaves are distinctly labeled by species. Rooted triplets are one of the most important inputs for constructing rooted phylogenetic trees. A rooted triplet is the simplest possible rooted tree that contains information and explains the biological relation between three species.

The problem of constructing a rooted phylogenetic tree that contains the maximum number of input triplets is a maximization problem and is known as the maximum rooted triplets consistency (MRTC) problem. MRTC problem is an NP-hard problem, so there is no any polynomial exact solution for it. The goal is to introduce a new efficient method to solve MRTC.

In this research, a new algorithm called CBTH, is introduced innovatively for MRTC problem with the goal to improve the consistency of input rooted triplets with the final rooted phylogenetic tree.

In order to show the efficiency of CBTH, the CBTH is compared with TRH on biological data. According to our knowledge, TRH is one of the best methods for MRTC problem on rooted triplets that are obtained from biological data. The Experimental results show that CBTH outperforms TRH based on rooted triplet consistency parameter in the same time order.

The introduced method (CBTH) solve MRTC problem with high performance without increasing time complexity compared to the other state of the art algorithms.

Clove oil is known for its medicinal properties. The mechanism of anti-cancer properties of Syzygium aromaticum were investigated by mathematical modelling on the genome scale with metabolomics using 1H Nuclear Magnetic Resonance spectroscopy on Raji cells.

An integrative analysis correlated the metabolites identified by 1HNMR and genes with the detected pathways.

Raji cells treated with clove oil were collected and sent for 1HNMR spectroscopy and the spectra analyzed by MATLAB and Human Metabolome Database for metabolite identification. Pathway and topology analysis was implemented using the genes and metabolites in the integrative analysis of Metaboanalyst software.

50% inhibitory concentration of clove oil was 50 μg/ml and the model anticipated 74 genes with differentiating metabolites being some amino acids, cholesterol and fucose.

The integrative study predicted that the anti cancer mechanism of clove oil involves novel enzymes, as likely drug targets, 24-dehydrocholesterol reductase and 7-dehydrocholesterol reductase in cholesterol biosynthesis, dehydrofolate reductase in one carbon metabolism and serine palmitoyl-transferase long chain in sphingolipid biosynthesis.

The biooxidation of ferrous iron has a great potential for the regeneration of ferric iron, in operations such as bioleaching, bioremediation. Many natural inorganic materials were investigated for use as supports immobilizing Acidithiobacillus ferrooxidans. The waste chalcopyrite is another natural inorganic material of which particles are easy to prepare from the leached out ore heaps and the source is abundant.

The aim of this work is to investigate several characteristics of the particles of waste ore that determines possibility of use as supports for immobilization of Acidithiobacillus ferrooxidans in the packed-bed bioreactor.

Acidithiobacillus ferrooxidans-1333 stored in Korean Centre for Culture Collection was used. The supports were prepared by sieving the particles of 5~30 mm in size out from the waste chalcopyrite ore heap. The cells were immobilized by the successive batch culture method and oxidation rate of the bioreactor was investigated in the continuous flow mode.

The cell density of Acidithiobacillus ferrooxidans-1333 immobilized on the particles of waste chalcopyrite was 2.71×108 cells g-1 and the highest oxidation rate of the packed-bed bioreactor was 3.65g.L-1.h-1. Oxidation rate of the bioreactor was less influenced by the concentration of ferrous and ferric iron in the input solution as well as by the aeration rate and dilution rate than other materials mentioned in other previous works.

The waste chalcopyrite particle is efficient support material for immobilization of Acidithiobacillus ferrooxidans with comparable or superior characteristics to natural inorganic support materials reported before.

Lignin is the largest natural aromatic polymer in nature and is also a unique aromatic-based biopolymer, accounting for nearly 30% of the earth’s organic carbon. Generally, lignin is regarded as waste and is mainly used as a lowvalue fuel that is burned to generate heat and energy to solve the problem of biomass waste; for this obstacle of lignin, highly efficient biodegradation plays a critical role in developing an environmentally friendly technique for lignin biotransformation.

This study intends to isolate and purify several microbial strains from nature. It also explores how their lignin degradation is able to enhance the biodegradation and recycling of biomass and the reclamation of lignin in wastewater from pulp and paper mills.

Lignin-degrading microbial strains were isolated from soil using medium containing sodium lignosulphonate as the sole carbon source. They were then screened by aniline blue and guaiacol plate, and then the best strain was chosen and identified. The conventional one-factor method was used to optimize various parameters that affect lignin’s degradation ability.

The strain possessing the highest lignin biodegradation ability was identified and denominated as Aspergillus flavus F-1. After optimization, the maximum degradation rate of lignin, 44.6% within 3 days, was obtained at pH 7.0, 30 ℃, 2.5 g·L-1 ammonium sulfate, 2 g·L-1 lignin and 0.5 g·L-1 glucose. The results show the LiP and Lac secreted from Aspergillus flavus F-1 played the main role in the degradation of lignin.

One microbial strain, Aspergillus flavus F-1, was successfully isolated with a lignin-degrading ability that can cut the lignin into fragments. This provides a promising candidate for the transformation and utilization of crop waste biomass for various industrial purposes.

Kallar Kahar lake, Punjab, Pakistan is a rich source of phytoplankton which can be used for biofuel production. This study was conducted to investigate the presence of different microalgae species present in this lake and their possible utilization for bioenergy production. The crude culture was examined under microscope. Isolation of the identified species was carried out by using serial dilution and colony picking methods. Isolated strains were evaluated by investigating their biomass productivity, salinity resistance and auto-flocculation ability. Four different microalgae species (Chlorella, Scenedesmus, Oscillatoria and Spirulina) were identified in the crude sample. The experimental results indicated that, among the four isolated strains, the Oscillatoria species showed highest biomass productivity (4.2 g.L) and Scenedesmus showed comparatively higher salt resistance. Scenedesmus also showed great potential of auto-flocculation as around 70 % of its cells sediment within 5 h without addition of any external flocculating agent. The lipid content in the isolated strains has also been carried out using Soxhlet extraction. Four different microalgae strains have been found in Kallar Kahar lake that reflected good biomass productivity and are capable of auto-flocculation.

Glyphosate is a non-selective systemic herbicide with a broad spectrum of weed control that inhibits a key enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, in the shikimate pathway.

Isolation and analysis of the epsps (aroA) gene responsible for glyphosate-tolerance in bacteria from Roundupcontaminated soils was the aim of this study.

Sampling was done from the soil of the gardens which were heavily contaminated by Roundup herbicide and then bacterial screening was performed in the presence of high concentrations of glyphosate. The genus of bacterium was identified via molecular methods such as 16S rRNA sequencing. The aroA gene of this bacterium (aroAHA-09) was isolated using the primers designed-upon specific regions of aroA genes available in NCBI GenBank database. The PCR product was cloned, sequenced and subcloned into pET28a as an expression vector and transferred into E. coli strainBL21(DE3). The cells were inoculated in liquid M9 minimal medium containing IPTG and different concentrations of glyphosate.

The genus of bacterium was identified as Pseudomonas sp. strain HA-09. The isolated aroAHA-09 gene from this bacterium was approximately 2.2 kb in size. Bioassay of E. coli expressing this gene showed high tolerance to glyphosate(up to 300 mM).

The aroAHA-09 gene could be considered as a novel and efficient candidate for development of glyphosatetolerant crop plants.

Use of avermectin B1b as anthelmintic and insecticidal agent has increased to protect soil and for enhanced crop production. Enhanced production of avermectin B1b was obtained from mutant strain of Streptomyces avermitilis 41445. Modeling of mutant strain S. avermitilis 41445 UV 45(m) 3 growth and avermectin B1b production is therefore required for optimization during fermentation process. Kinetics of intracellular avermectin B1b production was studies in shake flask culture during submerged fermentation. Mathematical models based upon Logistic and Piret Equations have been used to investigate the kinetics of avermectin B1b production and substrate utilization from S. avermitilis 41445 UV 45(m)3. Effect of carbon sources (glucose, maltose, lactose, potato starch, soluble corn starch, and wheat starch), pH (6.0, 6.5, 7.0, and 7.5), agitation speed (150, 200, and 250 rpm) on microbial growth and product formation were evaluated. Maximum avermectin B1b production (420.02mg.L-1 and cell biomass (31.74 g.L-1) were obtained in media having potato starch as carbon substrate at adjusted medium pH 7.5 and 250 rpm agitation speed. Maximum specific growth rate (µmax), growth associated avermectin B1b production coefficient (α) and non-growth associated avermectin B1b production coefficient (β) obtained were 0.16h-1, 0.0 mg.g-1 and 3.5 mg.g-1.h-1 respectively. From above results we can conclude that avermectin B1b production is non-growth associated process.

Sensitive detection of Maize Iranian mosaic virus (MIMV) in its insect vector, Laodelphax striatellus is essential for effective forecast and control of this viral disease. Three methods of ELISA, RT-PCR and IC-RT-PCR were compared regarding their sensitivity for detection of MIMV in the single planthopper with a series of various dilutions. To detect MIMV from a single insect vector, the sensitivity of three methods including ELISA, RT-PCR and IC-RT-PCR was evaluated. Compared to the other methods, the IC-RT-PCR showed more sensitivity and detected virus at least at the 1:60 dilution. While, both ELISA and RT-PCR methods could detect up to the 1:20. The results reported herein showed that IC-RT-PCR is a sensitive and simple method to detect MIMV from a single insect vector with high efficiency and reliability. These findings might be useful in the prediction of viral disease incidence in host plants and this method can also be effective to detect other viruses in their insect vectors.

Dynamic light scattering (DLS) and electron microscopy (EM) are the most practical techniques for nanoparticles (NPs) characterization. However, the impediments which involved the sample preparation method lead to failure in provided results of mentioned device analysis. These problems will be intensifying, if the examined samples are the soft nanocarriers such as organic ones or biological samples.

In order to achieve the appropriate results from DLS and EM analysis, an optimized protocol was introduced by this research which would prepare samples with high degree of quality and accuracy.

Morphological analysis of prepared polymeric nanocarriers (micelles, nanogels) by this protocol were done. Filtration, dilution and sonication as three crucial and effectiveness steps of sample preparation were assessed through DLS data and EM images.

This research has tried to introduce a facile method with novelty of simplicity and rapidity. These triple steps could improve the quality of morphological data. The obtained results indicated that sample preparation methods have the most effective factors on sample size distribution and homogeneity of desired samples.

The suggested optimized preparation method will be helpful for all soft nanomaterial’s samples.