Publications by authors named "Alireza Ghassempour"

75 Publications

Detection of structural and conformational changes in ALS-causing mutant profilin-1 with hydrogen/deuterium exchange mass spectrometry and bioinformatics techniques.

Metab Brain Dis 2021 Jul 24. Epub 2021 Jul 24.

Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran.

The hydrogen/deuterium exchange (HDX) is a reliable method to survey the dynamic behavior of proteins and epitope mapping. Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) is a quantifying tool to assay for HDX in the protein of interest. We combined HDX-MALDI-TOF MS and molecular docking/MD simulation to identify accessible amino acids and analyze their contribution into the structural changes of profilin-1 (PFN-1). The molecular docking/MD simulations are computational tools for enabling the analysis of the type of amino acids that may be involved via HDX identified under the lowest binding energy condition. Glycine to valine amino acid (G117V) substitution mutation is linked to amyotrophic lateral sclerosis (ALS). This mutation is found to be in the actin-binding site of PFN-1 and prevents the dimerization/polymerization of actin and invokes a pathologic toxicity that leads to ALS. In this study, we sought to understand the PFN-1 protein dynamic behavior using purified wild type and mutant PFN-1 proteins. The data obtained from HDX-MALDI-TOF MS for PFN-1 and PFN-1 at various time intervals, from seconds to hours, revealed multiple peaks corresponding to molecular weights from monomers to multimers. PFN-1/Benzaldehyde complexes identified 20 accessible amino acids to HDX that participate in the docking simulation in the surface of WT and mutant PFN-1. Consistent results from HDX-MALDI-TOF MS and docking simulation predict candidate amino acid(s) involved in the dimerization/polymerization of PFN. This information may shed critical light on the structural and conformational changes with details of amino acid epitopes for mutant PFN-1s' dimerization, oligomerization, and aggregation.
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http://dx.doi.org/10.1007/s11011-021-00763-yDOI Listing
July 2021

Two Dimensional Anion Exchange-Size Exclusion Chromatography Combined with Mathematical Modeling for Downstream Processing of Foot and Mouth Disease Vaccine.

J Chromatogr A 2021 Apr 15;1643:462070. Epub 2021 Mar 15.

Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran. Electronic address:

The production of high-quality purified virus particles in high quantities for vaccine preparation requires a scalable purification procedure in the downstream step. A purification scheme based on combined strong anion-exchange and size exclusion chromatography (2D-AEC-SEC) was developed for the production of non-structural protein-free foot and mouth disease vaccine, and the whole procedure was accomplished with 77.9% virus yield. Additionally, a mathematical modeling and a simulation approach based on a plate model of chromatography were developed and matched with the experimental chromatography data to improve prediction of retention behavior and save time in the development of the downstream scale-up method. The purified pooled virus fraction obtained from the final polishing step had a purity higher than 85% based on analytical size exclusion analysis. Moreover, more than 90.1% of residual DNA (rDNA) was removed from the purified vaccine. The analysis of purified virus particles by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS), high performance size exclusion chromatography (HP-SEC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and transmission electron microscopy (TEM) provided clear evidence of purity and demonstrated that the final product is structurally spherical, intact particles qualified for formulation as a vaccine product.
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http://dx.doi.org/10.1016/j.chroma.2021.462070DOI Listing
April 2021

In silico studies reveal structural deviations of mutant profilin-1 and interaction with riluzole and edaravone in amyotrophic lateral sclerosis.

Sci Rep 2021 03 25;11(1):6849. Epub 2021 Mar 25.

Department of Pharmacology and Toxicology, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, AR, 72205, USA.

This study aimed to investigate four of the eight PFN-1 mutations that are located near the actin-binding domain and determine the structural changes due to each mutant and unravel how these mutations alter protein structural behavior. Swapaa's command in UCSF chimera for generating mutations, FTMAP were employed and the data was analyzed by RMSD, RMSF graphs, Rg, hydrogen bonding analysis, and RRdisMaps utilizing Autodock4 and GROMACS. The functional changes and virtual screening, structural dynamics, and chemical bonding behavior changes, molecular docking simulation with two current FDA-approved drugs for ALS were investigated. The highest reduction and increase in Rg were found to exist in the G117V and M113T mutants, respectively. The RMSF data consistently shows changes nearby to this site. The in silico data described indicate that each of the mutations is capable of altering the structure of PFN-1 in vivo. The potential effect of riluzole and edaravone two FDA approved drugs for ALS, impacting the structural deviations and stabilization of the mutant PFN-1 is evaluated using in silico tools. Overall, the analysis of data collected reveals structural changes of mutant PFN-1 protein that may explain the neurotoxicity and the reason(s) for possible loss and gain of function of PFN-1 in the neurotoxic model of ALS.
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http://dx.doi.org/10.1038/s41598-021-86211-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994392PMC
March 2021

Venom Gland Mass Spectrometry Imaging of Saw-Scaled Viper, , at High Lateral Resolution.

J Am Soc Mass Spectrom 2021 Apr 16;32(4):1105-1115. Epub 2021 Mar 16.

Institute of Inorganic and Analytical Chemistry, Justus Liebig University, 35392 Giessen, Germany.

The snake venom gland is the place for the synthesis, storage, and secretion of a complex mixture of proteins and peptides, i.e., the venom. The morphology of the gland has been revealed by classical histology and microscopic studies. However, knowledge about the gland's cellular secretory and functional processes is still incomplete and has so far been neglected by the omics disciplines. We used autofocusing atmospheric-pressure matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) to investigate endogenous biomolecular distributions in the venom glands of the saw-scaled viper, , employing different sample preparation methods. Fresh-freezing and formalin-fixation were tested for the gland to obtain intact tissue sections. Subsequently, MSI was conducted with 12 μm pixel resolution for both types of preparations, and the lateral distributions of the metabolites were identified. Experiments revealed that lipids belonging to the classes of PC, SM, PE, PS, PA, and TG are present in the venom gland. PC (32:0) and SM (36:1) were found to be specifically located in the areas where cells are present. The snake venom metalloprotease inhibitor pEKW (/ 444.2233) was identified in the venom by top-down LC-MS/MS and localized by MALDI-MSI in the gland across secretory epithelial cells. The peptide can inhibit the venom's enzymatic activity during long-term storage within the venom gland. With a high degree of spectral similarities, we concluded that formalin-fixed tissue, in addition to its high ability to preserve tissue morphology, can be considered as an alternative method to fresh-frozen tissue in the case of lipid and peptide MS imaging in venom gland tissues.
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http://dx.doi.org/10.1021/jasms.1c00042DOI Listing
April 2021

Mass Spectrometry: A Powerful Method for Monitoring Various Type of Leukemia, Especially MALDI-TOF in Leukemia's Proteomics Studies Review.

Crit Rev Anal Chem 2021 Jan 26:1-28. Epub 2021 Jan 26.

Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Center, Cairo, Egypt.

Recent success in studying the proteome, as a source of biomarkers, has completely changed our understanding of leukemia (blood cancer). The identification of differentially expressed proteins, such as relapse and drug resistance proteins involved in leukemia by using various ionization sources and mass analyzers of mass spectrometry techniques, has helped scientists find better diagnosis, prognosis, and treatment strategies. With the aid of this powerful analytical technique, we can investigate the qualification/quantification of proteins, protein-protein interactions, post-translational modifications, and find the correlation between proteins and their genes with the hope of finding the missing parts of the successful therapy puzzle. In this review, we followed different MS sources and analyzers which used for monitoring various type of leukemia, then focused on MALDI-TOF MS as a quick and reliable method for studying proteins. Due to several review published for other techniques, the present review is the first work in this field. Also, by classifying more than 400 proteins, we have found 42 proteins are involved in two or three different stages of leukemia. Finally, we have suggested six specific biomarkers for AML, one for ALL, three biomarkers with a role in the etiology of leukemia and 13 markers with the potential for further studies.
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http://dx.doi.org/10.1080/10408347.2021.1871844DOI Listing
January 2021

Integrating Top-Down and Bottom-Up Mass Spectrometric Strategies for Proteomic Profiling of Iranian Saw-Scaled Viper, , Venom.

J Proteome Res 2021 01 22;20(1):895-908. Epub 2020 Nov 22.

Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Giessen 35392, Germany.

Saw-scaled or carpet vipers (genus ) are considered to cause a higher global snakebite mortality than any other snake. (ECS) is a widely distributed snake species, also found across the thirteen provinces of Iran, where it is assumed to be responsible for the most snakebite envenomings. Here, we collected the Iranian specimens of ECS from three different geographically distinct populations, investigated food habits, and performed toxicity assessment and venom proteome profiling to better understand saw-scaled viper life. Our results show that the prey items most commonly found in all populations were arthropods, with scorpions from the family Buthidae particularly well represented. LD (median lethal dose) values of the crude venom demonstrate highly comparable venom toxicities in mammals. Consistent with this finding, venom characterization via top-down and bottom-up proteomics, applied to both crude venoms and size-exclusion chromatographic fractions, revealed highly comparable venom compositions among the different populations. By combining all proteomics data, we identified 22 protein families from 102 liquid chromatography and tandem mass spectrometry (LC-MS/MS) raw files, including the most abundant snake venom metalloproteinases (SVMPs, 29-34%); phospholipase A2 (PLA2s, 26-31%); snake venom serine proteinases (SVSPs, 11-12%); l-amino acid oxidases (LAOs, 8-11%), C-type lectins/lectin-like (CTLs, 7-9%) protein families, and many newly detected ones, e.g., renin-like aspartic proteases (RLAPs), fibroblast growth factors (FGFs), peptidyl-prolyl cis-trans isomerases (PPIs), and venom vasodilator peptides (VVPs). Furthermore, we identified and characterized methylated, acetylated, and oxidized proteoforms relating to the PLA2 and disintegrin toxin families and the site of their modifications. It thus seems that post-translational modifications (PTMs) of toxins, particularly target lysine residues, may play an essential role in the structural and functional properties of venom proteins and might be able to influence the therapeutic response of antivenoms, to be investigated in future studies.
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http://dx.doi.org/10.1021/acs.jproteome.0c00687DOI Listing
January 2021

Protein ion yield enhancement in matrix-assisted laser desorption/ionization mass spectrometry after sample and matrix low-pressure glow discharge plasma irradiation.

Rapid Commun Mass Spectrom 2021 Jan;35(2):e8964

Department of Physics, Shahid Beheshti University, G.C., Evin, Tehran, Iran.

Rationale: Plasma-assisted ionization is widely used in mass spectrometry; in this study, a low-pressure glow discharge is introduced as a new method to improve the detection of large proteins, and bovine serum albumin (BSA) is used as a protein model. The treatment of analyte, matrix, and the matrix/analyte mixture is evaluated under optimal conditions.

Methods: Low-pressure radio-frequency capacitively coupled plasma (RF-CCP) treatment is utilized in the sample preparation step of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to enhance the protein MALDI ion signal. Plasma treatment can be an effective tool for enhancing the non-covalent binding of the analyte with the matrix, incorporation of the analyte into the matrix, production of matrix/analyte crystals, and analyte protonation through plasma activation, resulting in an improved MALDI ion signal.

Results: Fourier-transform infrared (FTIR) spectroscopy allows us to distinguish between the functional groups of plasma-treated and control samples. In addition, optical emission spectroscopy (OES) determines the plasma species, and zeta potential analysis characterizes the potential difference between plasma-treated and control samples before MALDI-TOF MS analysis. Plasma-treated BSA can provide a five-times enhancement of ion intensity. The combination of the plasma-treated analyte with the plasma-treated matrix leads to an increase in the ion intensity by a factor of 14.

Conclusions: Low-pressure glow discharge plasma treatment greatly enhances MALDI ion signals, with a noticeable increase in incorporation, co-crystallization, protonation, and the concentration of the sample functional groups.
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http://dx.doi.org/10.1002/rcm.8964DOI Listing
January 2021

Mechanism of antibodies purification by protein A.

Anal Biochem 2020 11 20;609:113909. Epub 2020 Aug 20.

Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C, Evin, P.O.Box: 19835-389, Tehran, Iran. Electronic address:

Protein A, a major cell wall component of Staphylococcus aureus, is one of the first immunoglobulin-binding proteins that is discovered about 80 years ago. However, a great deal of development in both purification methods and application of antibodies in treatment have been done. There are many publications based on the untargeted (size exclusion, ion exchange and hydrophobic interactions) and targeted (affinity) methods by scientists in academic/industry groups. In this review, we have focused on the study of both native and engineered Protein A to understand its mechanism in the purification of antibodies. What domain of Protein A dose interact with antibody? Where are contact regions? What is the non-covalent interaction mechanism of Protein A and antibody? Does alkaline condition, in the washing step, influence on antibody structure and activity? On the other hand, the immobilization of Protein A on various sorbents such as agarose, silica, polysaccharide, polymers, and magnetic nanoparticles have investigated. Also, the application of Protein A as biosensor for detection of the antibody is discussed. We have tried to find interesting and stimulating answers to all these questions.
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http://dx.doi.org/10.1016/j.ab.2020.113909DOI Listing
November 2020

Enantioseparation of mandelic acid on vancomycin column: Experimental and docking study.

Chirality 2020 11 14;32(11):1289-1298. Epub 2020 Aug 14.

Medicinal Plants and Drug Research Institute, Shahid Beheshti University, Evin, Tehran, Iran.

So far, no detailed view has been expressed regarding the interactions between vancomycin and racemic compounds including mandelic acid. In the current study, a chiral stationary phase was prepared by using 3-aminopropyltriethoxysilane and succinic anhydride to graft carboxylated silica microspheres and subsequently by activating the carboxylic acid group for vancomycin immobilization. Characterization by elemental analysis, Fourier transform infrared spectroscopy, solid-state nuclear magnetic resonance, and thermogravimetric analysis demonstrated effective functionalization of the silica surface. R and S enantiomers of mandelic acid were separated by the synthetic vancomycin column. Finally, the interaction between vancomycin and R/S mandelic acid enantiomers was simulated by Auto-dock Vina. The binding energies of interactions between R and S enantiomers and vancomycin chiral stationary phase were different. In the most probable interaction, the difference in mandelic acid binding energy was approximately 0.2 kcal/mol. In addition, circular dichroism spectra of vancomycin interacting with R and S enantiomers showed different patterns. Therefore, R and S mandelic acid enantiomers may occupy various binding pockets and interact with different vancomycin functions. These observations emphasized the different retention of R and S mandelic acid enantiomers in vancomycin chiral column.
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http://dx.doi.org/10.1002/chir.23273DOI Listing
November 2020

Conjugation of Single-Chain Variable Fragment Antibody to Magnetic Nanoparticles and Screening of Fig Mosaic Virus by MALDI TOF Mass Spectrometry.

Anal Chem 2020 08 15;92(15):10460-10469. Epub 2020 Jul 15.

Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran.

The ability of mass spectrometry for discrimination between protein and peptide masses which are unique to specific pathogens provides an accurate and fast method for the detection of different types of pathogens, especially viruses. Capsid proteins are specific to each virus and can be used as a biomarker for detection of this pathogen. On the other hand, single-chain variable fragment (scFv) antibodies have been recently used to enhance the accuracy of immunoassay techniques. So conjugation of mass spectrometry and scFv antibody provides a very accurate and fast method for the detection of viruses. In this report, for the first time, we have immobilized scFv antibody of fig mosaic virus (FMV) on the magnetic nanoparticles (MNPs) to extract the virus capsid protein from complex biological media and subsequently identified this protein through both its intact molecular mass and peptide mass fingerprint (PMF) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).
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http://dx.doi.org/10.1021/acs.analchem.0c01119DOI Listing
August 2020

A click tyrosine zwitterionic stationary phases for hydrophilic interaction liquid chromatography.

J Chromatogr A 2020 Jun 13;1621:461045. Epub 2020 Mar 13.

Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran. Electronic address:

New zwitterionic (ZIC) stationary phases (SPs) are synthesized with the click and conventional bonding of tyrosine to silica gel. Infrared spectra and elemental analysis demonstrate the successful click and conventional bonding of this ZIC group on silica particles by the surface coverage including 2.36 and 0.75 µm m, respectively. Given the above-mentioned explanation, the present study evaluated the retention mechanism and chromatographic manners of polar compounds on these new materials under hydrophilic interaction liquid chromatography (HILIC) conditions. Based on the results, the Click-Tyrosine Stationary Phase provided good HILIC characteristics when it was applied to separate phenolic compounds, amino acids, alkaloids, and nucleobases compared to bare silica gel SP and even conventional tyrosine SPs. Further, this new Click-Tyrosine-SP represented appropriate HILIC features and column efficiency (the theoretical plate number was up to 50,000 plates m for thebaine). Furthermore, the study investigated the effect of solute polarity (the number of the hydroxyl group of phenolic compounds) and hydrophobicity (the number of the side chain of aliphatic amino acids) on retention behaviors. Finally, some important factors were studied as the potential variables for guiding the retention behavior of the polar compound in HILIC condition including solvent composition, salt concentration, and the buffer pH of the mobile phase.
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http://dx.doi.org/10.1016/j.chroma.2020.461045DOI Listing
June 2020

Data from quantitative serum proteomic analysis after laparoscopic gastric plication.

Data Brief 2019 Aug 30;25:104077. Epub 2019 May 30.

Department of Health Science and Technology, Aalborg University, Denmark.

Bariatric surgery is an effective treatment for morbid obesity with a sustained weight loss and improvements in metabolic syndrome. We present a label free quantitative shotgun proteomics approach to analyze the serum proteome of obese people who underwent Laparoscopic Gastric Plication (LGP) as a new bariatric surgery. Pre-surgery serum samples of obese individuals were compared with the serum of the same subjects 1-2 months post-surgery (T1) and 4-5 months post-surgery (T2). The data provide a list of 224 quantifiable proteins with at least two unique peptides that were quantifiable in at least 70% of samples. Gene ontology biological processes and molecular functions of differentially regulated proteins between pre- and post-surgery samples were investigated using WebGestalt online tool. In addition, molecular networks of differentially abundant proteins were determined through Ingenuity Pathway Analysis (IPA) software. This report is related to the research article entitled "Serum proteome changes and accelerated reduction of fat mass after Laparoscopic Gastric Plication in morbidly obese patients" (Savedoroudi et al. [1]). Proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository through the identifier PXD010528.
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http://dx.doi.org/10.1016/j.dib.2019.104077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580113PMC
August 2019

Serum proteome changes and accelerated reduction of fat mass after laparoscopic gastric plication in morbidly obese patients.

J Proteomics 2019 07 3;203:103373. Epub 2019 May 3.

Department of Health Science and Technology, Aalborg University, Denmark. Electronic address:

Laparoscopic Gastric Plication (LGP) is a relatively new bariatric surgical procedure which no part of the stomach is removed. It is not clearly understood how LGP leads to fatty tissue reduction. We aimed to investigate the impact of LGP on serum proteome and understand molecular mechanisms of LGP-induced weight loss post-surgery. A Prospective observational study of 16 obese individuals who underwent LGP was performed. A Label-free quantitative shotgun proteomics approach was used to compare serum proteome of subjects before surgery with serum of the same individuals 1 to 2 months post-surgery (T1) and 4 to 5 months post-surgery (T2). The proteome analysis revealed that 48 proteins were differentially regulated between pre-surgery and T1, and seven proteins between pre-surgery and T2 of which six proteins were shared between the two timepoints. Among differentially regulated proteins, four proteins (SRGN, FETUB, LCP1 and CFP) have not previously been described in the context of BMI/weight loss. Despite few differences following LGP, most regulated serum proteins are in accordance with alternative weight loss procedures. Pathway analysis revealed changes to lipid- and inflammatory pathways, including PPARα/RXRα, LXR/RXR and FXR/RXR activation, especially at T1. At T2, the pathways related to inflammation and immune system are most affected. SIGNIFICANCE: Among the available clinical therapies for morbid obesity, bariatric surgery is considered as the most effective approach to achieve long-term weight loss, alongside a significant improvement in metabolic syndrome. However, very little is known about the underlying mechanism associated with significant weight loss post-surgery. Understanding such mechanisms could lead to development of safer non-surgical weight loss approaches. We here present the first analysis of the impact of LGP on the serum proteome, to bring new insights into the underlying molecular mechanism. Our findings indicate that LGP has a comprehensive systemic effect based on the blood serum proteome profile which might account for accelerated reduction of fat mass after surgery, thus, food restriction is not the only reason for weight loss following this unique surgical approach. As secretory regions of the stomach are preserved in LGP and it is associated with minimal physiological and anatomical changes, the findings are of high importance in the field of bariatric surgery and weight loss.
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http://dx.doi.org/10.1016/j.jprot.2019.05.001DOI Listing
July 2019

The effects of salicylic acid and glucose on biochemical traits and taxane production in a Taxus baccata callus culture.

Plant Physiol Biochem 2018 Nov 11;132:271-280. Epub 2018 Sep 11.

Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Tehran, Iran. Electronic address:

The combined use of elicitors can be an effective way to increase the production of secondary metabolites (SMs) in plant cell, tissue and organ cultures. This study investigated the effects of a salicylic acid (SA) pretreatment and different glucose levels on the growth, biochemical traits and taxane production in a Taxus baccata callus culture. For this purpose, after a pretreatment with SA (5 μM), three-month-old calli were cultured on B5 medium fortified with different concentrations of glucose (0, 0.5, 1, 2 and 3%), and they were compared with calli cultured on a B5 medium supplemented only with glucose. When the calli were harvested at 21 days, their fresh weight (g), dry weight (g) and cell viability (%) had decreased significantly (p < 0.05) with the higher glucose concentrations. The glucose treatment increased the hydrogen peroxide (HO) and malondialdehyde (MDA) content, and caused oxidative stress in treated tissues. The lower HO content and oxidative stress was associated with an increased antioxidant enzyme activity in the SA-pretreated samples, which resulted in less membrane damage and improved growth and cell viability under the glucose treatment compared to the control. By reducing the activity of phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO), the SA pretreatment reduced the production and oxidation of phenolic compounds under the glucose treatment; this decrease was associated with less browning of tissues and higher viability. Increases in taxol (5.1-fold) and total taxanes (3.5-fold) in the SA-pretreated calli cultured on the medium containing 2% glucose, compared to the control, indicated that the two treatments had a significant effect on taxane production in the T. baccata callus culture.
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http://dx.doi.org/10.1016/j.plaphy.2018.09.013DOI Listing
November 2018

Changes in biophysical characteristics of PFN1 due to mutation causing amyotrophic lateral sclerosis.

Metab Brain Dis 2018 12 10;33(6):1975-1984. Epub 2018 Sep 10.

Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran.

Single amino acid mutations in profilin 1 (PFN1) have been found to cause amyotrophic lateral sclerosis (ALS). Recently, we developed a mouse model for ALS using a PFN1 mutation (glycine 118 to valine, G118V), and we are now interested in understanding how PFN1 becomes toxically lethal with only one amino acid substitution. Therefore, we studied mutation-related changes in the PFN1 protein and hypothesized that such changes significantly disturb its structure. Initially, we expressed and studied the purified PFN1 and PFN1 proteins from bacterial culture. We found that the PFN1 protein has a different mean residue ellipticity, as measured by far-UV circular dichroism, accompanied by a spectral shift. The intrinsic fluorescence of PFN1 showed a small fluctuation in maximum fluorescence absorption and intensity. Moreover, we examined the time course of PFN1 aggregation using SDS-PAGE, western blotting, and MALDI-TOF/TOF and found that compared with PFN1, PFN1 had an increased tendency to form aggregates. Dynamic light scattering data confirmed this, showing a larger size distribution for PFN1. Our data explain why PFN1 tends to aggregate, a phenotype that may be the basis for its neurotoxicity.
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http://dx.doi.org/10.1007/s11011-018-0305-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6230493PMC
December 2018

Development and modeling of two-dimensional fast protein liquid chromatography for producing nonstructural protein-free food-and-mouth diseases virus vaccine.

J Chromatogr B Analyt Technol Biomed Life Sci 2018 Oct 21;1096:113-121. Epub 2018 Aug 21.

Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, Tehran, Iran. Electronic address:

Concerns for the use of non-purified or incompletely purified inactivated foot-and-mouth disease (FMD) vaccine, like difficulties for differentiation vaccinated from infected animals, can be a motivation in order to develop methods based on size exclusion chromatography (SEC). In this study, a two dimensional size exclusion chromatography (2D-SEC) system was successfully constructed using two different SEC column media to achieve a high-throughput purification system for the cell culture-derived foot and mouth diseases virus (FMDV). A mathematical model was also utilized to predict and to get a better insight into the separation process. Column and the packing particles characteristics such as column void volume, total column volume, particle porosity and accessible particle porosity was acquired experimentally. Retention times and elution profile of two different molecules, blue dextran and bovine serum albumin, were used for evaluating the capability of SEC media for separating two critical impurities (residual DNA (rDNA) and non-structural protein (NSP)) from active ingredient of vaccine (FMDV particle). Experiments were carried out with two different commercial columns (XK 26/60) and (XK 16/100) and with four different packing media superdex 200 prep grade, sephacryl S-500 HR, Sephacryl S-400 HR and Sephacryl S-300HR. The mathematical model was first validated by experimental chromatographic data of different SEC media and was then used to propose the best 2D-SEC system for downstream processing of the FMDV vaccine. The loading capacity of the constructed 2D-SEC sample was increased to 12.5% of total column volume and the purity of the final product was more than 90%. The entire purification process was performed with 77% FMDV recovery and 79.1% virus yield. Based on the high-performance size exclusion chromatography (HPSEC), the purity of the final NSP-free FMDV was about 90% and over 94.6% of host cell DNA was removed. Analyses of the purified FMDV by HPSEC, transmission electron microscopy (TEM) and dynamic light scattering (DLS) indicated that the final product had spherical shape with mean size about 30 nm and their structure remained intact.
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http://dx.doi.org/10.1016/j.jchromb.2018.08.014DOI Listing
October 2018

A perspective view of top-down proteomics in snake venom research.

Rapid Commun Mass Spectrom 2019 May 26;33 Suppl 1:20-27. Epub 2018 Sep 26.

Institute of Inorganic and Analytical Chemistry, Justus Liebig University Giessen, Germany.

The venom produced by snakes contains complex mixtures of pharmacologically active proteins and peptides which play a crucial role in the pathophysiology of snakebite diseases. The deep understanding of venom proteomes can help to improve the treatment of this "neglected tropical disease" (as expressed by the World Health Organization [WHO]) and to develop new drugs. The most widely used technique for venom analysis is liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based bottom-up (BU) proteomics. Considering the fact that multiple multi-locus gene families encode snake venom proteins, the major challenge for the BU proteomics is the limited sequence coverage and also the "protein inference problem" which result in a loss of information for the identification and characterization of toxin proteoforms (genetic variation, alternative mRNA splicing, single nucleotide polymorphism [SNP] and post-translational modifications [PTMs]). In contrast, intact protein measurements with top-down (TD) MS strategies cover almost complete protein sequences, and prove the ability to identify venom proteoforms and to localize their modifications and sequence variations.
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http://dx.doi.org/10.1002/rcm.8255DOI Listing
May 2019

A comparison and column selection of Hydrophilic Interaction Liquid Chromatography and Reversed-Phase High-Performance Liquid Chromatography for detection of DNA methylation.

Anal Biochem 2018 09 19;557:123-130. Epub 2018 Jul 19.

Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C. Evin, Tehran, Iran. Electronic address:

5-methylcytosine (5mC) is an epigenetic mark which has a profound effect on various fundamental processes in cells. In present study, at first Hydrophilic Interaction Liquid Chromatography (HILIC) was compared with Reversed-Phase High-Performance Liquid Chromatography (RP-HPLC) based on their selectivity (α), retention factor (k), and resolution (R) for cytosine (C) and 5mC nucleobases. We tried to justify the separation mechanism on the basis of mobile phase and solute polarity, structural characterization of solute and stationary phases, log Do/w, and pk under both modes. Then, these two modes were compared in order to select the best column for measurement of methylation level in two real samples with less analytical complexity (i.e. animal and bacteria) and a highly complex sample (i.e. plant), after chemical hydrolysis of DNA. In this favor, diol and cyano (CN) columns in HILIC mode as well as C and C in RP-HPLC were investigated. Optimum separation and the best validation parameters were obtained for CN column with Limit of Detection (LOD) of 1.4 pmol and Limit of Quantification (LOQ) of 4.8 pmol for 5mC. When the CN column was used in HILIC-UV procedure, separation of 5mC and C bases was achieved in all types of hydrolyzed DNA solutions.
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http://dx.doi.org/10.1016/j.ab.2018.07.013DOI Listing
September 2018

Resistant/susceptible classification of respiratory tract pathogenic bacteria based on volatile organic compounds profiling.

Cell Mol Biol (Noisy-le-grand) 2018 Jun 30;64(9):6-15. Epub 2018 Jun 30.

Pediatric Infections Research Center, Research Institute for Children Health, Shahid Beheshti University of Medical Sciences, Tehran, Iran.

Resistance to antibiotics is an emerging and growing threat. To address this threat, attempts are being made by researchers to identify the Volatile Organic Compounds (VOCs) of bacteria. It is believed that unique combinations could be found among the VOCs produced by each microorganism. The current study aimed to identify and compare the VOCs of antibiotic-resistant and standard strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. A polymer of divinylbenzene /carboxen /polydimethylsiloxane was applied for absorption of volatile compounds in headspace bacterial samples in form of a solid phase micro-extraction fiber holder. Gas chromatography-mass spectrometry technique was used for identification of volatile compounds. The analysis of the VOCs indicated that some VOCs appeared only in standard strains while others were common only among resistant strains. Exclusive VOCs to a specific strain were also detected. This study demonstrated that resistant strains of bacteria produced VOCs that were different from those of the standard strains. In addition, VOCs released by bacteria after passing the logarithmic growth phase showed no significant differences. The identification of VOCs can be a precise way to differentiate bacterial species, also it can be said that the VOCs produced by different pathogenic microorganisms can be the suitable biomarkers for their detection.
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June 2018

Initial study of three different pathogenic microorganisms by gas chromatography-mass spectrometry.

F1000Res 2017 10;6:1415. Epub 2017 Aug 10.

Department of Biology, Medicinal plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.

: Diagnoses  of  respiratory  tract  infections  usually happen  in  the  late  phase  of  the  disease  and  usually  result  in  reduction  of  the  pathogen  load after broad-spectrum  antibiotic  therapy,  but  not  in eradication of the pathogen.  The  development  of a  non-invasive,  fast,  and  accurate  method  to  detect  pathogens  has  always  been  of  interest  to  researchers  and  clinicians  alike.  Previous studies have shown that bacteria produce organic gases.  The  current  study  aimed  to  identify  the  volatile  organic  compounds  (VOCs)  produced  by three  respiratory  tract  pathogens,  including  ,   and  : The  VOCs  produced  were identified by gas chromatography-mass spectrometry (GC-MS), with  prior  collection  of  microbial  volatile  compounds  using  solid  phase  microextraction  (SPME)  fiber.  The volatile compounds were collected by obtaining bacterial headspace samples. : Results  showed  that  these  three  organisms  have  various  VOCs,  which  were  analyzed  under  different  conditions.  By ignoring common VOCs, some species-specific VOCs could be detected.  The most important VOC of was indole, also some important VOCs produced by   were 2,3-pentandione,  cis-dihydro-α-terpinyl  acetate,  1-decyne,  1,3-heptadiene,  2,5-dimethyl  pyrazine,  ethyl  butanoate  and  cyclohexene,4-ethenyl. Furthermore,  most  of the identified  compounds  by  are  alcohols. : The  detection  of  VOCs  produced  by  infectious  agents  maybe  the  key  to  make   a  rapid  and  precise  diagnosis  of  infection,  but  more  comprehensive  studies  must  be  conducted  in this  regard.
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http://dx.doi.org/10.12688/f1000research.12003.3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5760968PMC
August 2017

Cyclotides Isolated from an Ipecac Root Extract Antagonize the Corticotropin Releasing Factor Type 1 Receptor.

Front Pharmacol 2017 25;8:616. Epub 2017 Sep 25.

Center for Physiology and Pharmacology, Medical University of ViennaVienna, Austria.

Cyclotides are plant derived, cystine-knot stabilized peptides characterized by their natural abundance, sequence variability and structural plasticity. They are abundantly expressed in Rubiaceae, Psychotrieae in particular. Previously the cyclotide kalata B7 was identified to modulate the human oxytocin and vasopressin G protein-coupled receptors (GPCRs), providing molecular validation of the plants' uterotonic properties and further establishing cyclotides as valuable source for GPCR ligand design. In this study we screened a cyclotide extract derived from the root powder of the South American medicinal plant ipecac () for its GPCR modulating activity of the corticotropin-releasing factor type 1 receptor (CRFR). We identified and characterized seven novel cyclotides. One cyclotide, caripe 8, isolated from the most active fraction, was further analyzed and found to antagonize the CRFR. A nanomolar concentration of this cyclotide (260 nM) reduced CRF potency by ∼4.5-fold. In contrast, caripe 8 did not inhibit forskolin-, or vasopressin-stimulated cAMP responses at the vasopressin V receptor, suggesting a CRFR-specific mode-of-action. These results in conjunction with our previous findings establish cyclotides as modulators of both classes A and B GPCRs. Given the diversity of cyclotides, our data point to other cyclotide-GPCR interactions as potentially important sources of drug-like molecules.
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http://dx.doi.org/10.3389/fphar.2017.00616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627009PMC
September 2017

Optimization of throughput in semipreparative chiral liquid chromatography using stacked injection.

Chirality 2017 10 27;29(10):579-588. Epub 2017 Jul 27.

Phytochemistry Department, Medicinal Plants and Drugs Research Institute, Shahid Beheshti UniversityEvin, Tehran, Iran.

An interesting mode of chromatography for preparation of pure enantiomers from pure samples is the method of stacked injection as a pseudocontinuous procedure. Maximum throughput and minimal production costs can be achieved by the use of total chiral column length in this mode of chromatography. To maximize sample loading, often touching bands of the two enantiomers is automatically achieved. Conventional equations show direct correlation between touching-band loadability and the selectivity factor of two enantiomers. The important question for one who wants to obtain the highest throughput is "How to optimize different factors including selectivity, resolution, run time, and loading of the sample in order to save time without missing the touching-band resolution?" To answer this question, tramadol and propranolol were separated on cellulose 3,5-dimethyl phenyl carbamate, as two pure racemic mixtures with low and high solubilities in mobile phase, respectively. The mobile phase composition consisted of n-hexane solvent with alcohol modifier and diethylamine as the additive. A response surface methodology based on central composite design was used to optimize separation factors against the main responses. According to the stacked injection properties, two processes were investigated for maximizing throughput: one with a poorly soluble and another with a highly soluble racemic mixture. For each case, different optimization possibilities were inspected. It was revealed that resolution is a crucial response for separations of this kind. Peak area and run time are two critical parameters in optimization of stacked injection for binary mixtures which have low solubility in the mobile phase.
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http://dx.doi.org/10.1002/chir.22727DOI Listing
October 2017

Response surface methodology based on central composite design accompanied by multivariate curve resolution to model gradient hydrophilic interaction liquid chromatography: Prediction of separation for five major opium alkaloids.

J Sep Sci 2017 Sep 28;40(18):3602-3611. Epub 2017 Aug 28.

Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.

Hydrophilic interaction liquid chromatography on bare silica presents some benefits for analysis and purification of ionizable basic alkaloids. This mode was used to separate five major opium alkaloids: morphine, codeine, thebaine, papaverine, and noscapine. Central composite design based on response surface methodology was applied for experimental design, modeling, and optimization in a single-step gradient method. The main effects and their interactions (initial percentage of modifier, changing range of modifier in run time, pH of buffer, and its concentration) were investigated in 30 experiments. Multivariate curve resolution-alternating least squares, by resolving overlapped curves, helped in the accurate calculation of baseline resolution factors to be modeled and optimized more accurately. Then three crucial resolution factors besides elution time were modeled in quadratic and cubic equations and optimized. In addition to the four factors, five extra logarithmic, and nonlogarithmic factors extracted from the four factors to give nine factors overall were inspected on mechanism of retention. It was shown that a linear combination consist of four independence variables successfully describes morphinans retentivity in a single-step gradient method.
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http://dx.doi.org/10.1002/jssc.201700416DOI Listing
September 2017

Evaluation of hydrophilic interaction liquid chromatography stationary phases for analysis of opium alkaloids.

J Chromatogr A 2017 Aug 30;1511:77-84. Epub 2017 Jun 30.

Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12622, Egypt. Electronic address:

The separation of a mixture containing five major opium alkaloids, namely morphine, codeine, thebaine, noscapine and papaverine has been investigated in hydrophilic interaction liquid chromatography (HILIC) mode using five different stationary phases: bare silica, zwitterion, aminopropyl, diol and cyanopropyl. In order to propose the appropriate column for separation and purification, retention behaviors of the five natural opioids have been studied on mentioned HILIC stationary phases. The mechanism of separation in diverse HILIC media, based on the formation of water-rich layer on surface of the HILIC stationary phases and the physicochemical properties of opium alkaloids, such as pKa (acidic pK) and the octanol-water distribution coefficient (log Do/w) are discussed. Chromatographic responses including modified limit of detection LOD, signal to noise ratio (S/N), and defined modified R have considered for suggestion of the suitable column for quantitative/qualitative and preparative purposes. According to the obtained results, diol stationary phase is best suited for analytical chromatography, whereas bare silica and zwitterionic stationary phases are appropriate for preparative applications.
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http://dx.doi.org/10.1016/j.chroma.2017.06.072DOI Listing
August 2017

Untargeted metabolomic profiling of seminal plasma in nonobstructive azoospermia men: A noninvasive detection of spermatogenesis.

Biomed Chromatogr 2017 Aug 5;31(8). Epub 2017 Feb 5.

Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran.

Male factor infertility is involved in almost half of all infertile couples. Lack of the ejaculated sperm owing to testicular malfunction has been reported in 6-10% of infertile men, a condition named nonobstructive azoospermia (NOA). In this study, we investigated untargeted metabolomic profiling of the seminal plasma in NOA men using gas chromatography-mass spectrometry and advance chemometrics. In this regard, the seminal plasma fluids of 11 NOA men with TESE-negative, nine NOA men with TESE-positive and 10 fertile healthy men (as a control group) were collected. Quadratic discriminate analysis (QDA) technique was implemented on total ion chromatograms (TICs) for identification of discriminatory retention times. We developed multivariate classification models using the QDA technique. Our results revealed that the developed QDA models could predict the classes of samples using their TIC data. The receiver operating characteristic curves for these models were >0.88. After recognition of discriminatory retention time's asymmetric penalized least square, evolving factor analysis, correlation optimized warping and alternating least squares strategies were applied for preprocessing and deconvolution of the overlapped chromatographic peaks. We could identify 36 discriminatory metabolites. These metabolites may be considered discriminatory biomarkers for different groups in NOA.
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http://dx.doi.org/10.1002/bmc.3931DOI Listing
August 2017

Bypassing Protein Corona Issue on Active Targeting: Zwitterionic Coatings Dictate Specific Interactions of Targeting Moieties and Cell Receptors.

ACS Appl Mater Interfaces 2016 Sep 25;8(35):22808-18. Epub 2016 Aug 25.

Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences , Tehran, Iran.

Surface functionalization strategies for targeting nanoparticles (NP) to specific organs, cells, or organelles, is the foundation for new applications of nanomedicine to drug delivery and biomedical imaging. Interaction of NPs with biological media leads to the formation of a biomolecular layer at the surface of NPs so-called as "protein corona". This corona layer can shield active molecules at the surface of NPs and cause mistargeting or unintended scavenging by the liver, kidney, or spleen. To overcome this corona issue, we have designed biotin-cysteine conjugated silica NPs (biotin was employed as a targeting molecule and cysteine was used as a zwitterionic ligand) to inhibit corona-induced mistargeting and thus significantly enhance the active targeting capability of NPs in complex biological media. To probe the targeting yield of our engineered NPs, we employed both modified silicon wafer substrates with streptavidin (i.e., biotin receptor) to simulate a target and a cell-based model platform using tumor cell lines that overexpress biotin receptors. In both cases, after incubation with human plasma (thus forming a protein corona), cellular uptake/substrate attachment of the targeted NPs with zwitterionic coatings were significantly higher than the same NPs without zwitterionic coating. Our results demonstrated that NPs with a zwitterionic surface can considerably facilitate targeting yield of NPs and provide a promising new type of nanocarriers in biological applications.
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http://dx.doi.org/10.1021/acsami.6b05099DOI Listing
September 2016

Simple and Sensitive Quantification of Ghrelin Hormone in Human Plasma Using SBSE-HPLC/DAD-MS.

J Chromatogr Sci 2016 Oct;54(9):1652-1660

Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, Giza 12662, Egypt.

During a decade and more since its discovery, the emerging physiological roles of ghrelin in mammalian are increasingly being introduced, proposing a critical need for its quantification in biological milieu. Here in, a simple and sensitive single-step method for extraction and quantification of ghrelin in human plasma was developed and validated using stir bar sorptive extraction (SBSE) followed by high-performance liquid chromatography (HPLC) with diode array detection (DAD) coupled to mass spectrometry (MS). Influential parameters of SBSE procedure were optimized including extraction and desorption times of 45 and 30 min, respectively; pH of 4; no addition of salt. The sum of peak heights of three most intense selected ions in mass spectrum (844, 1125 and 1686 m/z) related to 4-, 3- and 2-fold-charged ions of ghrelin was used for quantification. Validation parameters containing linear dynamic range, limit of quantification and limit of detection were 0.02-80, 0.02 and 0.007 µg L-1, respectively, and calculated relative standard deviation for peak heights was 6.5% (0.7 µg L-1 standard solution). Mean recovery for ghrelin in spiked plasma samples was 96% ± 3. The efficiency of the SBSE-HPLC/DAD-MS procedure was proved by analysis of plasma samples from obese patients undergoing gastric plication surgery. The suggested methodology would contribute to simple and fast analysis of ghrelin levels in obesity and related diseases and also biochemical cycles in which gherlin is present.
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http://dx.doi.org/10.1093/chromsci/bmw125DOI Listing
October 2016

Recent developments in liquid chromatography-mass spectrometry analyses of ghrelin and related peptides.

Biomed Chromatogr 2017 Jan 21;31(1). Epub 2016 Aug 21.

Pharmaceutical and Medicinal Chemistry Department, Pharmaceutical and Drug Industries Research Division, National Research Center, Dokki, Giza, 12622, Egypt.

Profiling and monitoring concentrations of key hormones in body have long been critical aims in clinical therapy. As a crucial hormone, identification and quantification of ghrelin is a fundamental, often key, step in understanding human physiological mechanisms. Through the advances and improvements of different analytical techniques, ghrelin measurement is generally feasible, and the number of successful reports is progressively being increased with new aspects of selectivity, sensitivity and ease of use in various circumstances. Herein we discuss current chromatographic methods for sample collection, separation and a mass spectrometry method for detection and measurement of ghrelin and other proghrelin-derived peptides in biological metrics. We describe the most commonly applied analytical LC-MS procedures for determination of proghrelin-derived peptides and provide illustrative instances representing the state of the art. This review is intended for bioanalytical chemists or clinical researchers who are interested in this field of research.
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http://dx.doi.org/10.1002/bmc.3796DOI Listing
January 2017

Inhibition of the Aspergillus flavus Growth and Aflatoxin B1 Contamination on Pistachio Nut by Fengycin and Surfactin-Producing Bacillus subtilis UTBSP1.

Plant Pathol J 2016 Jun 1;32(3):209-15. Epub 2016 Jun 1.

Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran 19835-389, Iran.

In this study, the treatment of pistachio nuts by Bacillus subtilis UTBSP1, a promising isolate to degrade aflatoxin B1 (AFB1), caused to reduce the growth of Aspergillus flavus R5 and AFB1 content on pistachio nuts. Fluorescence probes revealed that the cell free supernatant fluid from UTBSP1 affects spore viability considerably. Using high-performance liquid chromatographic (HPLC) method, 10 fractions were separated and collected from methanol extract of cell free supernatant fluid. Two fractions showed inhibition zones against A. flavus. Mass spectrometric analysis of the both antifungal fractions revealed a high similarity between these anti-A. flavus compounds and cyclic-lipopeptides of surfactin, and fengycin families. Coproduction of surfactin and fengycin acted in a synergistic manner and consequently caused a strong antifungal activity against A. flavus R5. There was a positive significant correlation between the reduction of A. flavus growth and the reduction of AFB1 contamination on pistachio nut by UTBSP1. The results indicated that fengycin and surfactin-producing B. subtilis UTBSP1 can potentially reduce A. flavus growth and AFB1 content in pistachio nut.
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http://dx.doi.org/10.5423/PPJ.OA.11.2015.0250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4892817PMC
June 2016

A bioassay-guided fractionation scheme for characterization of new antibacterial compounds from Prosopis cineraria aerial parts.

Iran J Microbiol 2016 Feb;8(1):1-7

Department of Microbiology, School of Medicine, Tehran University of Medical Sciences, Poorsina street, Tehran, Iran.

Background And Objectives: Due to the importance of finding of new antibacterial agents, the antibacterial properties of Prosopis cineraria aerial parts were investigated using a bioassay guided fractionation scheme.

Materials And Methods: The organic extract was prepared via maceration in methanol, followed by the fractionation using n-hexane and ethyl acetate. The MICs of fractions were determined against some human pathogenic bacteria using broth micro-dilution assay. The primary characterization and identification of bioactive substance(s) was based on a bio-autographical method using HPTLC and flash chromatography in parallel with agar overlay assays. Finally the exact mass of effective compound(s) was determined by LC-MS.

Results: The best antibacterial activities were related to the ethyl acetate fraction. The effective antibacterial compound of the plant were 2 substances with molecular weight of 348 and 184 Dalton that inhibited the growth of assessed Gram positive bacteria with MIC values lower than 125 to 62.5 μg/ml synergistically.

Conclusion: Further analysis using nuclear magnetic resonance could reveal the exact structure of these two antibacterial substances. These 2 effective antibacterial compounds could be applied as lead compound for synthesis of new antibacterial agents.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4833735PMC
February 2016
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