Shiva Hemmati

Designing of Complex Multi-epitope Peptide Vaccine Based on Omps of Klebsiella pneumoniae: An In Silico Approach

Klebsiella pneumoniae is a hospital-acquired pathogen that leads to various infections. There is a great interest in developing an efficient Klebsiella vaccine and epitope-based vaccines may provide a hopeful choice. Outer membrane proteins (Omps) of gram-negative bacteria play an essential role in bacterial pathogenicity; therefore, they are ideal candidates for vaccine development against these pathogens. This study proposed an in silico approach by assembling consensus and high ranked epitopes of OmpW, OmpA, OmpF, OmpC and OmpX of K. pneumoniae and other virulence Enterobacteriaceae to induce multi-epitopic responses against these pathogens. Two vaccine constructs of discontinuous (conformational) B cell and linear CD4+ T cell epitopes of the Omps were designed. The three-dimensional structure of the B cell construct was successfully modeled and validated. Discontinuous B cell epitopes predictions of the B cell construct showed that the predicted epitopes are admissibly in common with total inserted discontinuous epitopes result in stimulation an efficient humoral immune response. In order to improve the immunogenicity, the size of the constructs was increased by multiplication of the epitopes and incorporation of sequence tags. Both constructs were found to be soluble, non-allergen and free of much posttranslational modification that could be effective on immunogenicity.

Accumulation of lignans by in vitro cultures of three Linum species.

Justicidin B, an arylnaphthalene lignan, has strong cytotoxicity on chronic myeloid and chronic lymphoid leukemia cell lines. The first report of the production of justicidin B in a Linum species concerned in vitro culture of Linum austriacum. Therefore, culture characterization and presence of arylnaphthalene-type lignans in calli and plantlets of Linum tenuifolium from section Linastrum, Linum bienne, and Linum glaucum from section Linum were studied. Seed germination of L. tenuifolium in the light and darkness was significantly higher (p < 0.05) than of L. bienne in the light and L. glaucum in the darkness. L. tenuifolium seedling length in the darkness was significantly higher (p < 0.01) than under light conditions. There were no significant differences in the calli and shoot biomass weight, number and length of shoots in three species over one month, while the shoot diameter of L. bienne was significantly different (p < 0.05) from that of L. glaucum. Justicidin B was detected in L. glaucum callus and plantlet cultures by HPLC/MS/UV-DAD and HPLC coupled with a photodiode array detector. This finding is important from pharmaceutical point of view and shows the chemosystematic relation between L. glaucum and L. austriacum and this method will be a powerful tool for detecting natural products in interested and endangered medicinal plants.

Justicidin B: A Promising Bioactive Lignan

Adverse effects and drug resistance to the current onchopharmacologicals have increased the demand for alternative novel therapeutics. We herein introduce justicidin B, an arylnaphthalen lignan isolated from different plant origins, especially Justicia, Phyllanthus, Haplophyllum and Linum species. This cyclolignan exhibits a wide array of biological properties ranges from piscicidal to antifungal, antiviral and antibacterial activities. Activity against Trypanosoma brucei makes justicidin B a potential antiprotozoal agent for the treatment of neglected tropical diseases. Pharmacological properties like antiplatelet, anti-inflammatory and bone resorption inhibition have been also attributed to justicidin B. This compound is a potent cytotoxic substance on several cell lines, especially chronic myeloid and chronic lymphoid leukemia. Pharmacological values, natural variation, as well as biotechnological production of justicidin B by plant cell, tissue and organ culture are also described in this review. Chemical characteristics and chromatographic methods to identify justicidin B and its biosynthetic pathway have been discussed. Different approaches to the total synthesis of justicidin B are compared. This review would shed light on the role of justicidin B as an intriguing natural compound and provides a chance to optimize conditions for industrial applications.

Linum mucronatum: Organ to Organ Lignan Variations

The percentage of podophyllotoxin (PTOX) and its congener lignans were measured by HPLC in Linum mucronatum ssp. mucronatum (Linaceae) fresh plant organs. The highest amounts of PTOX (0.595 ± 0.060% g/g dry wt) and 6-methoxypodophyllotoxin (MPTOX) (1.491 ± 0.125% g/g dry wt) were found in the plant sexual organs. Whereas, the highest levels of β-peltatin, 5′-demethoxy-MPTOX and yatein were found in not developed buds, petals and sepals, respectively.

Engineering Human Urate Oxidase: Towards Reactivating It as an Important Therapeutic Enzyme.

Urate oxidase is considered as an important therapeutic enzyme used to control hyperuricemia. In spite of widespread distribution in numerous (micro)organisms, active urate oxidase is absent in higher primates (humans and apes) due to gene mutations. Considering the therapeutic significance of urate oxidase, further understanding on the inactivation process of the enzyme during primate evolution is critical. This study, therefore, aims to express genetically modified human urate oxidase in the methylotrophic yeast Pichia pastoris. Accordingly, the genetically modified human urate oxidase was successfully expressed intracellularly and extracellularly under the control of an alcohol oxidase promoter and was subjected to the enzyme activity assay. The results demonstrated that reactivating the non-functional human urate oxidase gene fully or even moderately by simply replacing the premature stop codons is impossible. This finding confirms the idea that a number of successive loss-of-function missense mutations occurred during evolution, making higher primates functional uricase-deficit and vulnerable to hyperuricemic disorders.

Genome-wide identification, functional prediction, and evolutionary analysis of the R2R3-MYB superfamily in Brassica napus

R2R3-MYB transcription factors (TFs) have been shown to play important roles in plants, including in development and in various stress conditions. Phylogenetic analysis showed the presence of 249 R2R3-MYB TFs in Brassica napus, called BnaR2R3-MYB TFs, clustered into 38 clades. BnaR2R3-MYB TFs were distributed on 19 chromosomes of B. napus. Sixteen gene clusters were identified. BnaR2R3-MYB TFs were characterized by motif prediction, gene structure analysis, and gene ontology. Evolutionary analysis revealed that BnaR2R3-MYB TFs are mainly formed as a result of whole-genome duplication. Orthologs and paralogs of BnaR2R3-MYB TFs were identified in B. napus, B. rapa, B. oleracea, and Arabidopsis thaliana using synteny-based methods. Purifying selection was pervasive within R2R3-MYB TFs. Kn/Ks values lower than 0.3 indicated that BnaR2R3-MYB TFs are being functionally converged. The role of gene conversion in the formation of BnaR2R3-MYB TFs was significant. Cis-regulatory elements in the upstream regions of BnaR2R3-MYB genes, miRNA targeting BnaR2R3MYB TFs, and post translational modifications were identified. Digital expression data revealed that BnaR2R3-MYB genes were highly expressed in the roots and under high salinity treatment after 24 h. BnaMYB21, BnaMYB141, and BnaMYB148 have been suggested for improving salt-tolerant B. napus. BnaR2R3-MYB genes were mostly up regulated on the 14th day post inoculation with Leptosphaeria biglobosa and L. maculan. BnaMYB150 is a candidate for increased tolerance to Leptospheria in B. napus.

Bag Culture: A Method for Root-Root Co-Culture

A method named “bag culture” was developed for coculturing of Linum persicum (section Syllinum) and L. austriacum (section Linum) hairy roots. For this propose L. austriacum and L. persicum hairy root cultures were established using Agrobacterium rhizogenes in McCown medium. L. persicum hairy roots in bags (1 mm2 mesh) were successfully grown together with L. austriacum hairy roots. The amounts of podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (MPTOX) produced by L. persicum hairy root cultures were detected using HPLC. The results indicated that the amounts of both lignans and growth indexes of the two hairy roots decreased, that may be partly due to a competition between the two types of culture in using precursors of biosynthetic metabolites and the amount of culture medium which is available for each hairy root. However, MPTOX (0.17 g/100 g DW) and PTOX (0.02 g/100 g DW) levels of the L. persicum single culture in bag were significantly higher than of the other cultures which may be due to the immobilization effect of the bag.

Root cultures of linum species section Syllinum as rich sources of 6-methoxypodophyllotoxin.

Linum spp. from section Syllinum are promising for the production of aryltetralin lignans like podophyllotoxin (PTOX) and 6-methoxypodophyllotoxin (MPTOX). MPTOX is a PTOX congener that has cytotoxic activity comparable with PTOX. In this study root cultures of Linum Bungei from section Dasyllinum, L. strictum from section Linastrum, L. album, L. mucronatum ssp. mucronatum and L. nodiflorum from section Syllinum were established and their MPTOX levels were investigated in 1000 ml flasks. Root cultures of L. mucronatum ssp. mucronatum and L. nodiflorum were used to examine cell growth and production of MPTOX during a culture period of 36 days in 250 ml flasks. Considerable amounts of MPTOX in root cultures (1000 ml flasks) of L. album (6 mg/100 g DW), L. mucronatum ssp. mucronatum (770 mg/100 g DW) and L. nodiflorum (91 mg/100 g DW) were detected while it wasn’t detected in root cultures of L. Bungei and L. strictum. In time course experiments, the maximum amount of MPTOX in L. nodiflorum root culture was at day 16 with 480 mg/ 100 g DW and the maximum amount of MPTOX in L. mucronatum ssp. mucronatum root culture was at day 12 with 130 mg/100 g DW. The results showed that root cultures of Linum species from section Syllinum are rich sources of MPTOX and since this lignan has remarkable cytotoxic activity, it can be used as a precursor for the production of antitumor agents.

TAT-mediated intracellular delivery of carboxypeptidase G2 protects against methotrexate-induced cell death in HepG2 cells

ABSTRACT Side effects of methotrexate (MTX) especially hepatotoxicity limits clinical applications of this anticancer agent. Carboxypeptidase G2 (CPG2) is administrated for the treatment of elevated plasma concentrations of MTX. In this study, we have investigated the intracellular delivery of CPG2 fused to the transactivator transduction domain (TAT) and its protective effects against MTX‐induced cell death of HepG2 cells. We have observed that both native and denatured forms of the enzyme transduced into the HepG2 cells efficiently in a concentration and time‐dependent manner. The denatured protein transduced with higher efficiency than the native form and was functional inside the cells. MTX exposure significantly decreased HepG2 cell viability in a dose‐ and time‐dependent manner. The cell viability after 24 and 48h of incubation with 100&mgr;M MTX was reduced to 44.37% and 17.69%, respectively. In cells pretreated with native and denatured TAT‐CPG2 protein the cell viability was 98.63% and 86.31% after 24 and 48h, respectively. Treatment with MTX increased the number of apoptotic HepG2 cells to 90.23% after 48h. However, the apoptosis percentage in cells pretreated with native and denatured TAT‐CPG2 was 21.49% and 22.28%, respectively. Our results showed that TAT‐CPG2 significantly prevents MTX‐induced oxidative stress by decreasing the formation of ROS and increasing the content of glutathione (GSH) and catalase activity. Our finding indicates that both native and denatured TAT‐CPG2 strongly protect HepG2 cells against MTX‐induced oxidative stress and apoptosis. Hence, intracellular delivery of CPG2 might provide a new therapeutic strategy for protecting against MTX mediated cytotoxicity. HIGHLIGHTSMethotrexate treatment decreased viability of HepG2 cells.Carboxypeptidase G2 (CPG2) converts MTX to its non‐toxic metabolites.TAT‐CPG2 as a cell penetrating complex efficiently transduced into the cells.TAT‐CPG2 fusion protein strongly protects HepG2 cells against MTX‐induced cell death.TAT‐CPG2 provides a new strategy against MTX‐induced cell toxicity.

In silico analyses of several signal peptides for the excretory production of phenylalanine ammonialyase in Escherichia coli

T applied anaesthetics are employed in order to eliminate pain caused by needle insertion and injection, thus ameliorating patient compliance. Furthermore, they are devoid of symptoms of superficial trauma and local reaction. Therefore, the aim of this work was to develop a new formulation of lidocaine, proposed to improve its clinical effectiveness in topical anaesthesia in terms of both enhanced anaesthesia and a prolonged duration of action. For this purpose, we incorporated lidocaine in nanostructured lipid carriers (NLC). Particle size and zeta potential measurements, Fourier transform infrared spectroscopy and Raman spectroscopy were performed to characterize the NLC system. Furthermore, DSC and XRD measurements were conducted to investigate lipid crystallization which plays a very important role in the performance of NLC carriers. Additionally, membrane diffusion and penetration studies were completed in vitro and ex vivo, followed by measurements on skin hydration and transepidermal water loss in vivo. Our results lead us to the conclusion that the developed nanostructured lipid carrier is a promising vehicle for the topical delivery of lidocaine. The penetration of the NLC formulation was remarkable through heat separated epidermis after 24 hours, and the observed skin hydrating and occlusive effect also makes this formulation a favourable dermal carrier system.Statement of the Problem: Day by day, the demand for biotherapeutics and recombinant proteins is increasing. Herein, cytoplasmic expression in prokaryotic and eukaryotic hosts has been widely accepted. However, there are several obstacles in the large-scale production of recombinant proteins. Recombinant proteins might form inclusion bodies or be degraded by proteases. Endogenous proteins might also interfere with the folding of a recombinant secretory protein. These factors, as well as the complicated downstream purification process, will result in loss of protein yield. Moreover, the yield of recombinant protein is not only related to expression levels, but also to translocation efficiency. Thus, the translocation efficiency could be increased by using signal peptides. Phenylalanine ammonialyase (PAL), involved in the first step of the phenylpropanoid pathway, catalyzes the deamination of phenylalanine to cinnamate and ammonia. PALs are ubiquitous in plants and also commonly found in fungi; however, animal lacks it. They are of special interest in several medical and industrial applications, including preparation of low phenylalanine diet, treatment of phenylketonuria and certain neoplastic tumors. Although several methods have been applied in the production of PAL, the final titers of PAL are still low, thereby impeding considerable industrialization of this enzyme.Background/Aims: The recent introduction of computerized surveillance systems has promoted the monitoring of adverse drug reactions (ADRs), a feature that facilitates voluntary reports and enables prompt feedback. To investigate the causative agents and severity of ADRs that occurred in a single hospital, we analyzed the features of 980 ADRs that occurred over 14 months after developing a computerized ADR reporting system in Hallym Sacred Heart Hospital. Methods: ADR data collected prospectively from September 2007 to October 2008 by a computerized reporting system were analyzed. The World Health Organization-Uppsala Monitoring Center (WHO-UMC) criteria were used to determinate causality for each ADR. Results: The number of ADR cases reported voluntarily increased rapidly since the introduction of the computerized ADR reporting system. Of the 980 cases, antibiotics (34.5%) were the most common causative drugs, followed by analgesics such as tramadol and its compound (15.2%), radiocontrast media (7.0%), narcotics (5.9%), and nonsteroidal anti-inflammatory drugs (NSAIDs) (5.5%). Fifty-nine (6.0%) and 206 (21.0%) cases were classified as severe and moderate reactions, respectively. The mean age was older in patients with severe ADRs than in patients with non-severe ADRs. The most common clinical features were skin manifestations, such as pruritus, skin eruptions, and urticaria. Gastrointestinal symptoms including nausea, vomiting, and diarrhea were the second most frequently reported ADRs. Among antibiotics, first-generation cephalosporins were the most frequently reported causative drugs, followed by second-generation cephalosporins, penicillin/β-lactamase inhibitors, and third-generation cephalosporins. While 11.6% of ADRs related to penicillin/β-lactamase inhibitors were classified as severe, there was only one severe ADR (1.1%) for first-generation cephalosporins. Most ADRs were reported equally in men and women, although female cases constituted aboutDietary supplement market is growing in Turkey. Local and multinational companies develop and introduce many new products for Turkish dietary supplements market. However, in marketing literature, a few academic researchers could be found regarding Turkish consumers’ behavior about dietary supplements. Understanding the awareness and utilization rates of nutritional support products is important at the point of designing strategies for producers, the state and related organizations. This general exploratory research was carried out in 60 different pharmacies located in different regions of Turkey (Mediterranean, Black Sea, Eastern Anatolia, Southeastern Anatolia, Central Anatolia Region). Face-to-face interviews were conducted with 253 people aged 18 years and over from September 2017 to February 2018 (5 months). The data collected by the questionnaire includes the factors that affect the use of nutritional support products by respondents in the study, the recognition and consumption rates, the product content and how regularly they use it. Respondents were found to use products for immunity (21.03%) at the beginning of their nutritional support products usage. Omega 3 fish oil (26.98%), multivitamins (17.86%) and CoQ10 (7.94%) were the most preferred products. The rate of natural supplements usage by individuals who thought that use of natural supplements is crucial, is around %71.43.. We believe this study will lead to more detailed research on healthy lifestyle market in the future.T aim of the present study was to establish the relation that exists between the striatal dopamine levels and urine dopamine content in hemiparkinsonism rat model. 20 Wistar rats were used and were randomized into two groups as follows: a) control group and b) lessoned injured group induced by (6-OHDA). All animals were re-tested on the same battery of motor tests that before lesion. The rotation test behavior test was assessed and striatal DA levels and urine DA were determined by HPLC, motor behavior fine tests were done and finally immunohistochemical (Hir+) striatum was done. We found a positive correlation between the dopamine levels in the striatum and the content dopamine in urine of rats (control vs. 6-OHDA group). Respect motor performance, the 6-OHDA group showed a significant fine motor impairment (grasp and advance) vs. control group (p<0.01). Immunostaining for tyrosine hydroxylase (TH) expression revealed no TH-immunoreactive (THir) neurons in any 6-OHDA animals vs. control group (p<0.01). Positive correlation between the dopamine levels in the striatum and the content dopamine in urine could be talking also, about a major proportion of urinary dopamine could be derived from the renal decarboxylation of circulating dopa and not dopaminergic system disturbance. The other hand, alterations of a forelimb motor function in rats could be only due to more vulnerability of striatal dopaminergic depletion and not to low periphery dopamine levels.Polyamidoamine (PAMAM) dendrimers as synthetic gene vectors are efficient gene delivery systems. In this study, a kind of α-cyclodextrin-PAMAM conjugates polymer (Cy D-G1) was synthesized as a gene delivery vector. Based on ~1H NMR detectation, about 6.4 PAMAM-G1 molecules was grafted onto an α-CD core. Agarose gel electrophoresis revealed that Cy D-G1 could efficiently bind with DNA to condense them into nano-scale particles, which showed a similar binding capacity of PEI-25 K. Besides, it could protect DNA from DNase I degradation in a low N/P ratio. When N/P ratio in the CyD-G1/DNA polyplex was 40, the average particle size of CyD-G1/DNA polyplex was about 120 nm, and zeta potential was +21 mV. This polyplex could maintain its particle size in serum-containing solution within 360 min. In comparison with PEI-25 K carrier, CyD-G1 showed low cytotoxicity in various cell lines. Cell transfection results showed that CyD-G1 efficiently delivered DNA into cells at N/P = 80 compared with Lipofectamine 2000 and PEI-25 K. Unlike Lipofectamine 2000 and PEI-25 K, in serum-containing test condition, CyD-G1/DNA polyplex could maintain the transgene activities. The results of confocal laser scanning microscopy indicated that most DNA entered into cell nuclei within 4 h, and this phenomenon was consistent with the results calculated by flow cytometry. Taken together, CyD-G1 showed good transgene activities and the gene delivery vector could be used not only in vitro but also in vivo.