Study the Effect of Calendula officinalis Extract Loaded on Zinc Oxide Nanoparticle Cream in Burn Wound Healing.

The skin, the body's largest organ, acts as a protective barrier against pathogens and environmental damage. Skin burns can result from heat, chemicals, friction, or electricity. Nanoscience has recently been utilized to create ointments and creams for burns. Zinc oxide nanoparticles are crucial due to their antimicrobial and antioxidant properties. In this study, a cream containing nanoparticles was loaded with calendula extract, and its ability to promote tissue healing was investigated in Wistar rats with skin burns. The zinc oxide nanoparticles were chemically synthesized and loaded with calendula extract. The morphology and physicochemical properties of the nanoparticles were confirmed by SEM, ZETA size, XRD, and FTIR assays. The MTT technique was employed to assess the cream's impact on fibroblast growth. The antimicrobial activity of the nanoparticles was investigated against Pseudomonas using the MIC method. Real-time PCR was used to determine the expression of the Bax and Bcl-2 genes in aeruginosa. The results showed that zinc oxide nanoparticles at high concentrations increased the proliferation of the fibroblast cells. Histopathological studies showed granulation and epithelialization of the tissue without any hemorrhage or tissue infection during the first days of treatment with this cream. The animal models treated with the cream showed an increase in Bcl-2 gene expression and a decrease in Bax expression. We concluded that zinc oxide nanoparticles loaded with calendula extract have a practical effect in healing burn wounds due to their unique antibacterial properties of zinc oxide nanoparticles and their anti-inflammatory and wound-healing effects. The synergistic effect of these two substances significantly improved the healing process. This newly developed cream can be introduced as a successful and viable treatment option in burn wounds.

Evaluation of basil (Ocimum basilicum) accessions under different drought conditions based on yield and physio-biochemical traits.

BACKGROUND: Basil is one of the most famous herbs, which has broad usage as a fresh vegetable and therapeutic and pharmaceutical services. The main abiotic stress limiting basil production globally is drought. As a result, appropriate drought screening-which effectively separates high-yielding but drought-sensitive genotypes from drought-tolerant genotypes-is necessary for the optimal selection of high-yielding basil cultivars under drought stress conditions. So, a split plot experiment with three replications based on a completely randomized design were carried out in a pot under field conditions for this investigation. Water levels (full irrigation or control, moderate stress, and severe stress) were assigned as main plots, while 22 basil accessions were given as sub-plots. In this study, leaf yield as well as physio-biochemical traits had measured on accessions. RESULTS: Our results revealed large variation in yield, essential oil (%), protein, proline, chlorophyll, total phenol and flavonoids traits across the 22 accessions. The percentage of leaf yield reduction in moderate drought stress than normal conditions showed that G1 (-6.5%), G17 (-7.05%), G20 (-9.01%), and G12 (-10.9%) accessions had the least changes, respectively. Although in severe drought stress than normal conditions, the G1 (-32.01%), G12 (-33.12%), G4 (-33.24%), G7 (-34.11%), and G17 (-34.93%) accessions had the least amount of change in plant leaf yield, respectively. Furthermore, the highest yield reduction occurred in moderate and severe stress conditions in G18 (-25.36%) and G8 (-42.98%) accessions, respectively. Cluster analysis based on the ward method in both conditions (moderate and severe drought conditions) placed the accessions in three groups, and accessions were identified as tolerant, whose average traits in that group were higher than the total average. The principal component analysis also showed that in moderate drought conditions, the first two components explained about 95.28% of the total variation, while in severe drought conditions, these two components explained about 96.37% of the total variation. CONCLUSIONS: The different multivariate analyses (cluster analysis, PCA, mean comparison) were used to identify tolerant and sensitive accessions based on all traits. The accessions G3, G4, G6, and G7 were found to be tolerant to stress, while G10, G15, G16, and G20 were found to be sensitive to drought. These accessions are a useful step in producing drought-tolerant, high-yielding accessions and can be utilized in breeding programs for basil.

Bioinformatics and Structural Analysis of Antigenic Variation in the Hemagglutinin Gene of the Influenza A(H1N1)pdm09 Virus Circulating in Shiraz (2013 to 2015).

Circulating influenza A virus provided an excellent opportunity to study the adaptation of the influenza A(H1N1)pdm09 virus to the human host. Particularly, due to the availability of sequences taken from isolates, we could monitor amino acid changes and the stability of mutations that occurred in hemagglutinin (HA). HA is crucial to viral infection because it binds to ciliated cell receptors and mediates the fusion of cells and viral membranes; because antibodies that bind to HA may block virus entry to the cell, this protein is subjected to high selective pressure. In this study, the locations of mutations in the structures of mutant HA were analyzed and the three-dimensional (3D) structures of these mutations were modeled in I-TASSER. Also, the location of these mutations was visualized and studied using Swiss PDB Viewer software and the PyMOL Molecular Graphics System. The crystal structure of the HA from A/California/07/2009 (3LZG) was used for further analysis. The new noncovalent bond formations in mutant luciferases were analyzed via WHAT IF and PIC, and protein stability was evaluated in the iStable server. We identified 33 and 23 mutations in A/Shiraz/106/2015 and A/California/07/2009 isolates, respectively; some mutations are located on the antigenic sites of Sa, Sb, Ca1, Ca2, and Cb HA1 and the fusion peptide of HA2. The results show that with the mutation some interactions are lost and new interactions are formed with other amino acids. The results of the free-energy analysis suggested that these new interactions have a destabilizing effect, which needs confirmation experimentally. IMPORTANCE Due to the fact that the mutations that occurred in the influenza virus HA cause the instability of the protein produced by the virus and antigenic changes and the escape of the virus from the immune system, the mutations that occurred in A/Shiraz/1/2013 were investigated in terms of energy level and stability. The mutations located in a globular portion of the HA are S188T, Q191H, S270P, K285Q, and P299L. On the other hand, the E374K, E46K-B, S124N-B, and I321V mutations are located in the stem portion of the HA (HA2). The change V252L mutation eliminates interactions with Ala181, Phe147, Leu151, and Trp153 and forms new interactions with Gly195, Asn264, Phe161, Met244, Tyr246, Leu165, and Trp167 which can change the stability of the HA structure. The K166Q mutation, which is located within the antigenic site Sa, causes the virus to escape from the immune response.

Correlation between total phenolic and flavonoid contents and biological activities of 12 ethanolic extracts of Iranian propolis.

Propolis is a resinous substance produced by honey bees that is very popular as a natural remedy in traditional medicine. The current research is the first study on the biological properties of ethanolic extracts of propolis (EEP) from several different regions (12) of Iran. Total phenolic and flavonoid contents (TPC and TFC) of Iranian EEPs were variable between 26.59-221.38 mg GAE/g EEP and 4.8-100.03 mg QE/g EEP. The DPPH scavenging assay showed all the studied EEP samples, except for the sample with the lowest TPC and TFC (P6), have suitable antioxidant activity. All the EEPs inhibited both cholinesterase enzymes (acetylcholinesterase: AChE, butyrylcholinesterase: BuChE) but most of them exhibited a distinct selectivity over BuChE. Evaluation of the antibacterial activity of the EEP samples using four pathogenic bacteria (B. cereus, S. aureus, A. baumannii, and P. aeruginosa) demonstrated that the antibacterial properties of propolis are more effective on the gram-positive bacterium. Spearman correlation analysis showed a strong positive correlation between TPC and TFC of the Iranian EEPs and their antioxidant, anticholinesterase, and antibacterial activities. Considering that there is ample evidence of anticholinesterase activity of flavonoids and a significant correlation between the anticholinesterase activity of the studied Iranian EEPs and their total flavonoid content was observed, the interaction of 17 well-known propolis flavonoids with AChE and BuChE was explored using molecular docking. The results indicated that all the flavonoids interact with the active site gorge of both enzymes with high affinity. Summing up, the obtained results suggest that Iranian propolis possesses great potential for further studies.

Thiadiazole - A promising structure in design and development of anti-Alzheimer agents.

The design and development of effective multitargeted agents in treating Alzheimer disease (AD) has always been a hot topic in the field of drug discovery. Since AD is a multifactorial disorder, various key hidden players such as deficit of acetylcholine (ACh), tau-protein aggregation, and oxidative stress have been associated with the incidence and progress of AD. In pursuit of improving efficacy and expanding the range of pharmacological activities of current AD drugs, the molecular hybridization method is also used intensively. Five-membered heterocyclic systems such as thiadiazole scaffolds have previously been shown to have therapeutic activity. Thiadiazole analogs as an anti-oxidant compound have been known to include a wide range of biological activity from anti-cancer to anti-Alzheimer properties. The suitable pharmacokinetic and physicochemical properties of the thiadiazole scaffold have introduced it as a therapeutic target in medicinal chemistry. The current review portrays the critical role of the thiadiazole scaffold in the design of various compounds with potential effects in the treatment of Alzheimer's disease. Furthermore, the rationale used behind hybrid-based design strategies and the outcomes achieved through the hybridization of Thiadiazole analogs with various core structures have been discussed. In addition, the data in the present review may help researchers in the design of new multidrug combinations that may provide new options for the treatment of AD.

Antioxidant Compounds in the Treatment of Alzheimer's Disease: Natural, Hybrid, and Synthetic Products.

Alzheimer's disease (AD) which is associated with cognitive dysfunction and memory lapse has become a health concern. Various targets and pathways have been involved in AD's progress, such as deficit of acetylcholine (ACh), oxidative stress, inflammation, ß-amyloid (Aß) deposits, and biometal dyshomeostasis. Multiple pieces of evidence indicate that stress oxidative participation in an early stage of AD and the generated ROS could enable neurodegenerative disease leading to neuronal cell death. Hence, antioxidant therapies are applied in treating AD as a beneficial strategy. This review refers to the development and use of antioxidant compounds based on natural products, hybrid designs, and synthetic compounds. The results of using these antioxidant compounds were discussed with the given examples, and future directions for the development of antioxidants were evaluated.

Effects of synonymous mutations on kinetic properties and structure of firefly luciferase: Molecular dynamics simulation, molecular docking, RNA folding, and experimental study.

Although synonymous mutations have long been thought to lack striking results, a growing body of research shows these mutations have highly variable effects. In this study, the impact of synonymous mutations in the development of thermostable luciferase was investigated using a combination of experimental and theoretical approaches. Using bioinformatics analysis, the codon usage features in the Lampyridae family's luciferases were studied and four synonymous mutations of Arg in luciferase were created. An exciting result was that the analysis of kinetic parameters showed a slight increase in the thermal stability of the mutant luciferase. AutoDock Vina, %MinMax algorithm, and UNAFold Server were used to perform molecular docking, folding rate, and RNA folding, respectively. Here, it was assumed that in the region (Arg337) with a moderate propensity for coil, synonymous mutation altered the rate of translation, which in turn may lead to a slight change in the structure of the enzyme. According to the molecular dynamics simulation data, local minor global flexibility is observed in the context of the protein conformation. A plausible explanation is that this flexibility may strengthen hydrophobic interactions due to its sensitivity to a molecular collision. Accordingly, thermostability originated mainly from hydrophobic interaction.

In silico Validation of Pseudomonas aeruginosa Exotoxin A Domain I Interaction with the Novel Human scFv Antibody.

BACKGROUND: Pseudomonas (P.) aeruginosa is one of the leading causes of nosocomial infections. The pathogenicity of P. aeruginosa is related to its inherent antimicrobial resistance and the diverse virulence factors of this bacterium. Owing to the specific role of exotoxin A in P. aeruginosa pathogenesis, it is known as a promising therapeutic candidate to develop antibodies as an alternative to antibiotics. OBJECTIVE: The present study aimed to validate the interaction between a single-chain fragment variable (scFv) antibody identified from an scFv phage library against domain I exotoxin A by bioinformatic tools. METHODS: For this, several bioinformatics tools, including Ligplot, Swiss PDB viewer (SPDBV), PyMOL, I-TASSER, Gromacs, and ClusPro servers were used to evaluate the interaction of scFv antibody with P. aeruginosa exotoxin A. The I-TASSER server was utilized to predict the function and structure of proteins. The interaction of two proteins was analyzed using ClusPro tools. The best docking results were further analyzed with Ligplot, Swiss PDB viewer, and PyMOL. Consequently, molecular dynamics simulation was utilized to predict the stability of the secondary structure of the antibody and the binding energy of the scFv antibody to the domain I of exotoxin A. RESULTS: As a result, we demonstrated that data from computational biology could provide proteinprotein interaction information between scFv antibody/domain I exotoxin A and offers new insights into antibody development and therapeutic expansion. CONCLUSION: In summary, a recombinant human scFv capable of neutralizing P. aeruginosa exotoxin A is recommended as a promising treatment for infections caused by P. aeruginosa.

Epidemiology, Geographical Distribution, and Outcome Analysis of Patients with Electrical Burns Referred to Shiraz Burn Center, Shiraz, Iran during 2008-2019.

Background: Burn is one of the most significant injuries in industrial and developing societies and is one of the most important traumas leading to hospitalization. The aim of this study was to identify the epidemiology, geographical distribution, and outcome of electric burns in Fars province and to present the distribution map. Methods: In this descriptive-analytical study, the study population involved all electrical burn victims admitted to Amir al-Momenin and Ghotbeddin Hospitals from 2008 to 2019 in Fars province in the south of Iran. Data were analyzed using SPSS software version 22. Results: Among a total of 246 patients, the average age was 30.78 ± 11.07. The highest frequency among educational levels was among under-diploma patients (38.6%), and the majority were employed (87.4%). Also, most of the patients were from urban areas (70.3%). The majority of burn incidences occurred at the workplace (57.7%). Also, among the high voltage patients, 25 patients (30.9%) had an amputation, while among low voltage only 12 patients (16.2%) had an amputation. Non-surgical treatment was applied in 68 (28%) cases, while Escharotomy was performed in 28 (11.4%) patients. There was also a statistically significant association between burn voltage and amputation (P= 0.039). Conclusion: Based on our report, the rate of electrical burn injuries in Iran is still high, which underlines the need for stronger efforts in effective prevention, such as better public education and the establishment of strict regulations regarding the distribution and use of electricity.

Synthesis and Characterization of GO/ZIF-67 Nanocomposite: Investigation of Catalytic Activity for the Determination of Epinine in the Presence of Dobutamine.

In this study, we prepared graphene oxide (GO)/ZIF-67 nanocomposites. Therefore, GO/ZIF-67 nanocomposites were used as a modifier on a screen-printed electrode (GO/ZIF-67/SPE) for studying the electrochemical behavior of epinine in phosphate buffer saline (PBS) at pH 7.0 with voltammetry techniques. The GO/ZIF-67/SPE showed greater electrocatalytic activities than the bare SPE. As a result, the GO/ZIF-67/SPE was utilized for additional electrochemical examinations. The epinine concentration determination was in the range 9.0 × 10-8 M to 5.0 × 10-4 M, and the limit of detection (LOD) as well as the limit of quantification (LOQ) equaled 2.0 and 6.6 nM, respectively. From the scan rate study, the oxidation of epinine was found to be diffusion-controlled, and the simultaneous detection of epinine and dobutamine were well achieved with the differential pulse voltammetric (DPV) technique. Moreover, the stability and reproducibility of epinine at the GO/ZIF-67/SPE was studied, and the use of the GO/ZIF-67/SPE to detect epinine and dobutamine in real samples was furthermore successfully demonstrated.

Thermally stable and acidic pH tolerant mutant phytases with high catalytic efficiency from Yersinia intermedia for potential application in feed industries.

Heat- and pH-stable phytase efficiently hydrolyzes phytic acid. In this research, heat- and pH-stable mutant phytases, T83R, L287R, and T83R/L287R were generated by site-directed mutagenesis from Yersinia intermedia. After the induction and expression of recombinant wild-type and mutant phytases in E. coli BL21, the enzymes were purified using nickel sepharose affinity chromatography, and characterized kinetically and thermodynamically using spectroscopy methods. The mutants showed optimum activity at pH 5.15 and 55-61 °C. The catalytic efficiencies of T83R, L287R, T83R/L287R, and wild-type phytases were calculated to be 2941, 29346, 4906, and 6917 mmol/L-1s-1, respectively. Moreover, after the incubation of T83R, L287R, wild-type, and T83R/ L287R phytases at 100 °C for 1 h, the enzymes retained 22, 5, 4, and 2% of their initial activities, respectively. In addition, T83R, T83R/L287R, L287R, and wild-type phytases retained 82, 44, 16 as well as 11% of their initial activities after 1 h at pH 5.15, respectively. Among these mutants, T83R mutant showed 18% increase in thermal stability, 71% increase in pH stability, and +0.103 KJ/mole increase in ΔΔG, while the catalytic efficiency and ΔΔG value of L287R mutant increased by 4 times and +0.0903 KJ/mole, respectively. Thus, the mutants have the potential to be used in feed industries to increase the bioavailability of minerals while decreasing soil and water pollution.

Engineering of Ocriplasmin Variants by Bioinformatics Methods for the Reduction of Proteolytic and Autolytic Activities.

Background: Ocriplasmin has been developed for the induction of posterior vitreous detachment in patients with vitreomacular adhesion. At physiological pH, ocriplasmin is susceptible to autolytic and proteolytic degradation, limiting its activity duration. These undesirable properties of ocriplasmin can be reduced by site-directed mutagenesis, so that its enzymatic activities can be augmented. This study aimed to design ocriplasmin variants with improved biological/physicochemical characteristics via bioinformatics tools. Methods: This study was performed in Tabriz University of Medical Sciences, Tabriz, Iran, 2019. Through site-directed mutagenesis, three ocriplasmin variants were designed. Structural analysis was performed on the wild-type variant and the mutant variants using the Protein Interactions Calculator (PIC) server. The interactions between the S-2403 substrate and the ocriplasmin variants were studied by molecular docking simulations, and binding capability was evaluated by the calculation of free binding energy. The conformational features of protein-substrate complex systems for all the variants were evaluated using molecular dynamic simulations at 100 nanoseconds. Results: The structural analysis of ocriplasmin revealed that the substitution of threonine for alanine 59 significantly reduced proteolytic activity, while the substitution of glutamic acid for lysine 156 influenced autolytic function. The molecular docking simulation results indicated the appropriate binding of the substrate to the ocriplasmin variants with high-to-low affinities. The binding affinity of the wild-type variant for the substrate was higher than that between the mutant variants and the substrate. Simulation analyses, consisting of the root-mean-square deviation, the root-mean-square fluctuation, and the center-of-mass average distance showed a higher affinity of the substrate for the wild type than for the mutant variants. Conclusion: The mutational analysis of ocriplasmin revealed that A59T and K156E mutagenesis could be used for the development of a new variant with higher therapeutic efficacy.

Assessment of E. coli Expression System for Overexpression of Active Recombinant Ocriplasmin.

Purpose: Ocriplasmin (Jetrea TM) is a FDA approved recombinant enzyme utilized in the treatment of vitreomacular adhesion (VMA). This is a recombinant C-terminal fragment of human plasmin produced using yeast Pichia pastoris. Since ocriplasmin does not contain any Oor N-glycosylation or some other post-translational modifications, bacterial expression systems such as Escherichia coli could be considered as an economical host for recombinant expression. In the present study, we aimed to evaluate the efficiency of E. coli expression system for highlevel expression of recombinant ocriplasmin. Methods: The gene coding for ocriplasmin was cloned and expressed in E. coli BL21. The bacterial cells were cultured on large scale and the expressed recombinant protein was purified using Ni-NTA chromatography. Refolding of denatured ocriplasmin to active enzyme was carried out by the stepwise removal of denaturant. The identity of recombinant ocriplasmin was confirmed using western blotting and ELISA assays. The presence of the active ocriplasmin was monitored by the hydrolytic activity assay against the chromogenic substrate S-2403. Results: The final yield of E. coli BL21-produced ocriplasmin was approximately 1 mg/mL which was greater than that of P. pastoris. Using western blotting and ELISA assay, the identity of recombinant ocriplasmin was confirmed. The hydrolysis of chromogenic substrate S-2403 verified the functional activity of E. coli produced ocriplasmin. Conclusion: The results of this study indicated that E. coli could be used for high level expression of ocriplasmin. Although the recombinant protein was expressed as inclusion body, the stepwise refolding leads to the biologically active proteins.

Enhancement of Thermostability of Aspergillus flavus Urate Oxidase by Immobilization on the Ni-Based Magnetic Metal-Organic Framework.

The improvement in the enzyme activity of Aspergillus flavus urate oxidase (Uox) was attained by immobilizing it on the surface of a Ni-based magnetic metal-organic framework (NimMOF) nanomaterial; physicochemical properties of NimMOF and its application as an enzyme stabilizing support were evaluated, which revealed a significant improvement in its stability upon immobilization on NimMOF (Uox@NimMOF). It was affirmed that while the free Uox enzyme lost almost all of its activity at ~40-45 °C, the immobilized Uox@NimMOF retained around 60% of its original activity, even retaining significant activity at 70 °C. The activation energy (Ea) of the enzyme was calculated to be ~58.81 kJ mol-1 after stabilization, which is approximately half of the naked Uox enzyme. Furthermore, the external spectroscopy showed that the MOF nanomaterials can be coated by hydrophobic areas of the Uox enzyme, and the immobilized enzyme was active over a broad range of pH and temperatures, which bodes well for the thermal and long-term stability of the immobilized Uox on NimMOF.

Immobilization of Lepidium draba peroxidase on a novel Zn-MOF nanostructure.

In the present study, ultrasound irradiation was utilized to synthesize a novel zinc metal-organic framework (MOF). Scanning electron microscopic images, exhibited homogenous morphology with a nano-sized distribution of the Zn-MOF structure as also confirmed by X-ray diffraction patterns. Following, physical immobilization of Lepidium draba peroxidase (LDP) were optimized on the Zn-MOF in phosphate buffer (50 mM, pH 6.5), ratio amount of MOF/enzyme; 7/1 after shaking for 15 min at 25 °C, with high protein loading of 109.9 mg/g and immobilization yield of 93.3%. Immobilized enzyme (IE) exhibited more than 330% enhanced specific activity and also exhibited more than 150% specific affinity to its substrate (3,3',5,5'-tetramethylbenzidine) with respect to the free enzyme (FE). Optimum temperature of the IE was obtained at 20 °C while its was 25 °C for the FE, and thermostability of the IE augmented at temperature of 30 °C and 40 °C by the factors of 104 and 108% respectively. pH stability under neutral and basic condition and storage stability of the IE improved with respect to the FE as well as its structural stability (Tm; 73 °C for IE vs. 63 °C for FE). Furthermore, immobilization is accompanied with alteration on the enzyme structure as revealed by the intrinsic and extrinsic fluorescence spectra.

The Evaluation of tLyP-1-Bound Mda-7/IL-24 Killing Activity on a Liver Tumor Cell Line.

Background: The melanoma differentiation-associated gene-7 (Mda-7)/interleukin-24 (IL-24) is a tumor killing cytokine, the bystander effect of which can be enhanced through tethering to tumor homing peptides (THPs). Materials and Methods: After fusing tLyP-1, RGR, and buforin as THPs to Mda-7/IL-24, enzyme-linked immunosorbent assay (ELISA) was used to determine the secretion potency of the recombinant proteins. The killing potency of plasmids expressing IL-24, IL-24.tLyP1, IL-24.RGR, and buf.IL-24 were assessed, using MTT, Annexin/PI staining assays, as well as measuring the expression level of GADD-153 and BCL2-associated X (BAX) on Huh-7 cells. Three-dimensional structural analysis and protein-receptor interaction were also evaluated by modeling. Results: The ELISA result showed that contrary to IL-24.RGR and buf.IL-24, IL-24.tLyP-1 retained the secretion potency, similar to the native form. The viability assessments showed that IL-24 and IL-24.tLyP-1 had the most growth suppressive effects in comparison with the control group (p < 0.0001). Furthermore, IL-24 and IL-24.tLyP-1 had the highest apoptotic activity and significant upregulatory effect on the GADD-153 and BAX genes (p < 0.0003). The modeling showed that peptide modifications left no detrimental effect on IL-24 attachment to the cognate receptor. Conclusion: IL-24 can tolerate tLyP-1 peptide modification by retaining its secretion potency. Tethering tLyP-1 to IL-24 can induce more apoptosis than its modified versions by RGR or buforin.

Mutational Analysis of Ocriplasmin to Reduce Proteolytic and Autolytic Activity in Pichia pastoris.

BACKGROUND: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases. RESULTS: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced Kcat and Vmax values. An increase in their Km values, leading to a decreased catalytic efficiency (the Kcat/Km ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, Kcat and Vmax values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations. CONCLUSIONS: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

Design, synthesis and biological assessment of acridine derivatives containing 1,3,4-thiadiazole moiety as novel selective acetylcholinesterase inhibitors.

A novel series of acridine derivatives containing substituted thiadiazol-2-amine moiety was synthesized via multi-component condensation reaction of dimedone, aromatic aldehyde and 5-aryl-1,3,4-thiadiazol-2-amines in the presence of LaCl3 as a catalyst under solvent-free conditions. Anticholinesterase (AChE and BuChE) activity evaluation of the derivatives showed that all the derivatives are capable of inhibiting both enzymes and are highly selective towards AChE. Among them, the ability of 4i and 4d with respective IC50 values of 0.002 and 0.006 µM to inhibit AChE was higher than the reference compound tacrine (IC50 = 0.016 µM). The kinetics studies demonstrated that 4i and 4d inhibit AChE through a competitive/non-competitive mixed mechanism. The HEPG2 cell viability assay evidenced that 4i and 4d significantly exhibit lower hepatotoxicity compared with tacrine. Blind docking experiments performed on TcAChE (PDB ID: 2ACE) indicated that an unknown site is preferred for binding by all the derivatives over classic binding site of the enzyme, site 1 (CAS/PAS). Identification of the residues by protein structure alignment confirmed that this site is site 2 which was recently recognized as a new allosteric site of hAChE. The binding modes of 4i and 4d were also investigated using local docking studies on site 1 and site 2.

Genomic and protein structure analysis of the luciferase from the Iranian bioluminescent beetle, Luciola sp.

To date, two Iranian luciferase genes from the Lampyris turkestanicus and Lampyroidea maculata have been carefully studied. Here, we report the cloning and characterization of the gene and protein of luciferase enzyme from the beetle of an Iranian lampyrid species, Luciola sp. (Coleoptera-Lampyridae). In this study, a Luciola sp. firefly was collected from the Yasouj area of Iran and its luciferase gene sequence was cloned and characterized. The genomic DNA length for this luciferase was the 1950 bp that combined of seven exons and separated by six introns. The results of multiple sequence alignment show that this gene has the most similarity with DNA gene luciferase from the Hotaria unmunsana species. Further analysis determined accurately the location of these introns in the luciferase gene. However, the deduced amino acid sequences of the luciferase gene (548 residues) showed that this luciferase had 97.8% resemblance to luciferase from Lampyroidea maculata species. By in silico modeling of firefly luciferase in an I-TASSER server, the 3D structure of this enzyme was evaluated. The results of phylogenetic tree analysis display the close evolutionary relationship of this luciferase gene and luciferase gene from the Lampyroidea maculata and Hotaria unmunsana.

New Developments in Pichia pastoris Expression System, Review and Update.

BACKGROUND: Although Pichia pastoris is an outstanding host among conventional expression systems for production of recombinant proteins, a new interest has been emerged to this system due to the inherent advantages and new developments in this expression host. The potential for secretory and soluble expression of heterologous glycoproteins in P. pastoris proposed this system as a candidate for the production of complex eukaryotic proteins. METHODS: Several new developments have occurred in different areas related to P. pastoris expression system including hosts, vectors, glycosylation pattern and fermentation technology. Strain engineering using Crispr/Cas9 technology to produce human-like glycoproteins and protease deficient strains are two new areas of development with high importance. RESULTS: This review is dedicated to discuss the most important characteristics of P. pastoris with emphasis on new developments, especially in the field of glycoengineering, efficient expression vectors and promoters. CONCLUSION: New developments that occurred in the P. pastoris expression system converted this system to a versatile host for the production of complex proteins. This progress paved the way for several proteins to enter the clinical trials or industrial processes with this valuable expression host.