Papers by Malihe Keramati
AMB express, May 10, 2024
Tavianatou et al. 2019). Depending on the molecular size, HA fragments can influence cellular beh... more Tavianatou et al. 2019). Depending on the molecular size, HA fragments can influence cellular behavior in a different mode of action. Physiologically, low-molecularweight HAs (LMW-HA) (less than 200 kDa) or o-HA (Hyaluronan oligosaccharide) stimulate the proliferation and migration of endothelial cells (ECs) (angiogenic effect) (Deed et al. 1997; Sattar et al. 1994; West et al. 1985; West and Kumar 1989), whereas, high-molecularweight HAs (HMW-HA, n-HA) have an inhibitory effect (Kumar et al. 1989; West and Kumar 1988, 1991). There have been numerous recent reports that fragments of HA have different properties compared to the intact molecule (Baggenstoss et al. 2017). This phenomenon is attributed to the different manner of interaction with cell surface receptors, especially CD44 and RHAMM (Prosdocimi AMB Express
Research Square (Research Square), Aug 9, 2023
Background: The biological properties of hyaluronan (HA) are determined by its molecular weight (... more Background: The biological properties of hyaluronan (HA) are determined by its molecular weight (MW). Most natural extraction process of HA resulted in a polydisperse polymer. In the present study, the monodisperse LMW-HA was biosynthesized by truncated recombinant hyaluronan synthase (HAS) derived from Streptococcus equisimilis (SeHAS). The effects of t transmembrane domains truncation of SeHAS on productivity and speci c activity of enzymes, dispersity, MW, and biological activity of synthesized polymers were investigated. The recombinant HAS 123 (341 aa), HAS 23 (309 aa), and HAS Intra (263 aa) were constructed. The MW, structure, and titer of the polymer were studied by electrophoresis, FTIR spectroscopy and carbazole assay, respectively. The biological activity of HA polymers was assessed by cell proliferation and wound healing assays. Results: The speci c activities were calculated as 7.5, 6.8, 4.9, and 2.8 mg HA .mg protein-1 .min-1 for SeHAS, HAS 123 , HAS 23 , and HAS Intra , respectively. The results revealed SeHAS produced a polydispersed HMW-polymer (268 kDa), while HAS 123 and HAS 23 produced a polydisperse LMW-polymer (6.1, and 4.6 kDa, respectively). Interestingly, HAS Intra produced a monodisperse LMW-HA (1.9 kDa). Biological assessments indicated the LMW-HAs showed a dose-dependent proliferation activity on endothelial cells (ECs), whereas HMW-HAs exhibited an inhibitory effect. Also, LMW-HAs had the highest wound healing effect at 10 µg/mL, while by increasing in the concentration to 200 µg/mL, both LMW-and HMW-HAs postponed the wound healing recovery rate. Conclusion: The study indicated the transmembrane domains of SeHAS affect the titer, size, and dispersity of synthesized HA. These ndings open new insight into the rational engineering of SeHAS to produce sizede ned HA.
Journal of clinical microbiology and biochemistry technology, Jul 5, 2022
Proteases are the most important industrial enzymes which have attracted enormous attention due t... more Proteases are the most important industrial enzymes which have attracted enormous attention due to their vast variety and well-defined specificity. Microbial proteases are superior to other sources like plant and animal proteases because of their desired characteristics for biotechnological application. In this regard, Lysobacter enzymogenes is a rich source for the production of antibiotics and proteases. However, strain improvement in order to obtain overproduced microorganisms is always demanded at an industrial scale. Therefore, in the present study in order to enhance L. enzymogenes protease production, random mutagenesis was applied using N-methyl-N’-nitro-N-nitrosoguanidine (NTG) as a chemical mutagen. Random mutagenesis was conducted on L. enzymogenes suspension cultivated on nutrient broth using different concentrations of NTG (100, 150, and 200 µg/ml) for 20 and 40 minutes. The treated bacteria were cultivated on nutrient agar containing casein as a selective media. Primary and secondary screenings were performed by measuring the diameter of the casein hydrolysis zones in the isolated bacteria and the related supernatants, respectively. Finally, the unit of protease activity was quantified by Anson’s method of examining bacterial supernatants. Among the total of 30 isolated mutants, two mutants showed the highest level of extracellular proteolytic activity which showed 2.65 and 1.86 fold increments in contrast to the wild type, respectively. In general, the effect of mutagenesis by NTG can be emphasized to increase protease activity.
Archives of Microbiology, May 20, 2022
AMB Express
Hyaluronic acid (HA), composed of glucuronic acid (GlcUA) and N-acetyl glucoseamine (GlcNAc), is ... more Hyaluronic acid (HA), composed of glucuronic acid (GlcUA) and N-acetyl glucoseamine (GlcNAc), is a versatile biopolymer with high commercial value and innumerous physiological roles and pharmaceutical applications. The hasA gene has main role in HA biosynthesis by Streptococcus strain as a natural producer. The hasB and hasC genes are also mediate GlcUA precursor biosynthesis. In the present study, S. equisimilis hasA gene; B. subtilis tuaD and gtaB genes for GlcUA precursors enhancement, and vgb gene coding bacterial hemoglobin as an oxygen provider were used to construct the B. subtilis strain for HA production. RBSHA (hasA), RBSHA2 (hasA/tuaD/gtaB), and RBSHA3 (hasA/tuaD/gtaB/vgb) strains were developed and confirmed through genotype and phenotype analysis. After HA production and purification, FTIR spectroscopy confirmed the produced HA structures. HA assay showed the highest HA titer for RBSHA3 (2.1 ± 0.18 mg/ml) and then RBSHA2 (1.9 ± 0.03 mg/ml), and RBSHA (0.6 ± 0.14 mg/ml)....
World Journal of Microbiology and Biotechnology
Additional file 7: Table S3. Screening of 50 clones by fluorimetry 6 h after inoculation in a 96-... more Additional file 7: Table S3. Screening of 50 clones by fluorimetry 6 h after inoculation in a 96-well microplate. Ten clones with maximum and minimum fluorescent signals were selected for the enzyme activity assay.
Additional file 2: Table S2. Optimization of transferring different volumes of transformation sus... more Additional file 2: Table S2. Optimization of transferring different volumes of transformation suspension to obtain a single clone in each well of 96-well microplates. Data are represented from three independent experiments.
Additional file 4: Fig. S2. Fluorescence sensitivity measurements. Fluorescent signals were measu... more Additional file 4: Fig. S2. Fluorescence sensitivity measurements. Fluorescent signals were measured for 26 clones at the sensitivities of 60, 70, and 80. The only signals with a relative fluorescence unit (RFU) of
Additional file 10. Additional materials and methods for additional files 1 to 9.
Vaccine Research, 2018
Introduction: Serum bactericidal assay is the gold standard index of protection against Vibrio ch... more Introduction: Serum bactericidal assay is the gold standard index of protection against Vibrio cholerae. The outer membrane vesicles (OMVs) which are released during the bacterial growth have intrinsic immune stimulatory properties, based on their nature and composition. In this study, the induction of serum bactericidal activities in immunized BALB/c mice was determined using different vaccine regimens, using V. cholerae O1 (El Tor biotype) OMVs. Methods: A single clone of V. cholerae O1 (El Tor biotype) isolated during the 2005 outbreak in Iran, was used. A detergent-centrifugation procedure was used for OMVs production. Various vaccination regimens were inoculated into female mice via an oral route. The vaccine formulas included V. cholerae OMVs, killed whole cells of V. cholerae (WC), combination of WC-OMV and licensed cholera vaccine (Dukoral).The serum vibriocidal activity of mice sera was determined by measuring the complement-mediated lysis. Results: Electron microscopy of the purified OMVs from the isolated V. cholerae revealed the spherical-shaped vesicles of the size range 20-300 nm. In vitro reactivity of mice sera bactericidal capability against regimens of vaccination showed a significant immune response of antibody titers in comparison with negative control groups. Also, there was a significant increase in serum bactericidal titer of WC-OMV obtained from wild-type V. cholerae which had a satisfactory reactivity as Dukoral cholera vaccine. Conclusion: The results indicated that the combination of WC-OMV from the local strain is able to induce a high level of bactericidal antibody responses and it can be useful in optimization of the vaccine formula.
AMB Express
Design of experiment (DOE) is a statistical approach for designing, performing, and interpreting ... more Design of experiment (DOE) is a statistical approach for designing, performing, and interpreting a large set of data with the minimum number of tests. In our previous study, we developed a novel Hsp27 SILEX system for production of recombinant proteins. In the present study, we optimized indirectly the most effective factors including inoculation load, self-induction temperature, and culture media on autoinduction of staphylokinase (SAK) expression using RSM methodology and fluorometry. The expression level of SAK was assayed at different runs after 6 h incubation at 90 rpm. The results indicated all parameters significantly affect the SAK expression level (p < 0.05). The optimum expression condition was obtained with an inoculation load of 0.05, a temperature of 25 °C, and TB culture medium. The analysis of variance with a R2 value of 0.91 showed that a quadratic model well described this prediction (p < 0.05). Applying the optimized condition led to an approximately fourfold...
Additional file 3: Fig. S1. Time determination for fluorimetry.
Journal of Industrial Microbiology & Biotechnology, 2013
Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic strep... more Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SD...
Streptokinase (SK), a plasminogen activator (PA) that converts inactive plasminogen (Pg) to plasm... more Streptokinase (SK), a plasminogen activator (PA) that converts inactive plasminogen (Pg) to plasmin (Pm), is a protein secreted by groups A, C, and G <i>streptococci</i> (GAS, GCS, and GGS, respectively), with high sequence divergence and functional heterogeneity. While roles of some residual changes in altered SK functionality are shown, the underlying structural mechanisms are less known. Herein, using computational approaches, we analyzed the conformational basis for the increased activity of SK from a GGS (SKG132) isolate with four natural residual substitutions (Ile33Phe, Arg45Gln, Asn228Lys, Phe287Ile) compared to the standard GCS (SKC). Using the crystal structure of SK.Pm catalytic complex as main template SKC.μPm catalytic complex was modeled through homology modeling process and validated by several online validation servers. Subsequently, SKG132.μPm structure was constructed by altering the corresponding residual substitutions. Results of three independent MD ...
Iranian Journal of Microbiology, 2019
Cholera disease remains an important global health problem affecting 3-5 million subjects worldwi... more Cholera disease remains an important global health problem affecting 3-5 million subjects worldwide. Outer membrane vesicles (OMVs) have been found in a variety of Gram-negative bacteria and act as protective transport vesicles. The aim of this study was to evaluate Immune responses against Vibrio cholerae O1 El Tor clinical strain OMV and compare it with killed whole cell (KWC), complex of (KWC-OMV) as well as the internationally licensed oral cholera vaccine, Dukoral, in serum and intestinal secretions of mice. OMVs were prepared by using modified detergent-centrifugation procedure from V. cholerae O1 El Tor clinical strain from 2005 outbreak. The ultrastructure and content of OMVs were investigated via the Scanning Electron Microscopy (SEM) and SDS-PAGE analysis. Three doses of oral immunization were adjusted and total IgG and IgA in serum and intestinal secretion were measured by enzyme-linked immunosorbent assay (ELISA). Extracted OMVs from the V. cholerae were spherical vesicl...
Journal of Babol University of Medical Sciences, 2017
J Babol Univ Med Sci; 19(2); Feb 2017; PP: 20-5 Received: Jul 24 2016, Revised: Nov 26 2016, Acce... more J Babol Univ Med Sci; 19(2); Feb 2017; PP: 20-5 Received: Jul 24 2016, Revised: Nov 26 2016, Accepted: Jan 15 2017. ABSTRACT BACKGROUND AND OBJECTIVE: Whooping cough is an acute contagious human respiratory disease especially in infants and children and is one of the ten major causes of death from infectious diseases. Despite reducing the risk due to vaccination with killed bacteria pathogen (Bordetella pertussis), increased disease emerged in recent decades indicates re-emerging due caused by genetic diversity of bacteria and the insufficiency of current vaccines. Therefore, the design of new and effective vaccines based on common strains of the population is essential. In this study the inactive recombinant protein of most important bacterial antigens (pertosis toxin, PTX), the most commonly pathogenic strain in Iran, were produced on the basis of the new generation of non-cellular vaccines. METHODS: Bacterial strain used in this study to determine the genome and gene amplificatio...
The EpCAM (epithelial cell adhesion molecule) is a cell surface antigen over expressed in many ty... more The EpCAM (epithelial cell adhesion molecule) is a cell surface antigen over expressed in many types of epithelial cell cancers including colon, stomach, pancreas, lung, ovarian, and breast. So, it can be an attractive target for active and passive immunotherapy of cancers. ScFv (single chain fragment variable) fragment is a class of engineered antibodies in which the genes coding for the heavy (VH) and light chains (VL) of an immunoglobulin have been linked with a short flexible peptide linker. Inexpensive media, rapid growth rates, and relatively minimal laboratory set up make Escherichia coli (E. coli) a suitable host for expression of a large variety of recombinant proteins. Here, we assessed the effect of cultivation conditions on the level of expressed scFv against extracellular domain of EpCAM (EpEX) in E. coli. pET22b-antiEpEX-scFv was transformed into prepared E. coli Rosetta™(DE3) competent cells. To evaluate the effect of cultivation conditions on protein expression level...
Cloning and expression of streptokinase gene isolated from iranian streptoecci strains , Cloni... more Cloning and expression of streptokinase gene isolated from iranian streptoecci strains , Cloning and expression of streptokinase gene isolated from iranian streptoecci strains , بانک اطلاعاتی جامع اطلاع رسانی پایان نامه های کشور
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Papers by Malihe Keramati