Identification of Proteins Participating in the Cisplatin Resistance Following Treatment with Cisplatin in A2780 and A2780CP Ovarian Cell Lines

Kamyar Keshavarz Farajkhah, Maryam Tabarzad, Farshad Hosseini Shirazi, Mohammad Hassan Houshdar Tehrani

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-6 (e1)
https://doi.org/10.22037/tpps.v5i0.28873

Ovarian cancer is the most fatal gynecological cancer and the 8th most prevalent type of cancer in Iran. Chemotherapy regimen for the treatment of this type of cancer is mostly based on platinum agents and paclitaxel. The major problem during treatment with cisplatin is the appearance of acquired resistance in cancer cells in the first 6 months of therapy. Owing to inefficacy of second line regimens, it seems necessary to find out the molecular mechanisms of cisplatin resistance and find an efficient strategy against the resistant cancer cells. In this study, two ovarian cancer cell lines, A2780-sensitive and A2780CP (resistant to cisplatin) were evaluated. To acquire the protein expression profile, a culture of each line containing 1.5×107 cells was divided into the two groups of control and treatment cells. The treatment group cells were treated with cisplatin at pre-determined IC50 concentration for 6 hours. Then, the total proteins of 7×106 cells were extracted. The proteome of each group (20 μg) was used for subsequent separation of proteins by two-dimensional gel electrophoresis using 7 cm IPG strips. The results of protein pattern changes were analyzed by one-way ANOVA. At least 230 proteins were detected in each gel, from which, about 45 proteins were differentially expressed in each model of comparison. However, by considering the results of all models of analysis on the protein expression profile of two cell lines, three proteins were determined as the key players of resistance against cisplatin.


HIGHLIGHTS


  • The major problem during treatment with cisplatin is the appearance of acquired resistance in cancer cells.

  • The cisplatin- resistant and sensitive ovarian cell lines of A2780 were evaluated in this study.

  • Proteomics analysis was run using 2D gel electrophoresis after cisplatin treatment.

  • Three proteins were determined as the key players of resistance against cisplatin in A2780 cell line.

Effect of High Salinity on Mycosporine-Like Amino Acid Production in Desmodesmus sp.

Rouzbeh Gharib, Maryam Tabarzad, Tahereh Hosseinabadi

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-6 (e2)
https://doi.org/10.22037/tpps.v5i0.28876

Mycosporine like amino acids (MAAs) are UV absorbing compounds which are produced by a variety of organisms such as algae and cyanobacteria, in order to protect themselves from harmful UV irradiation. Thus, they can potentially be used as sunscreens in pharmaceutical and cosmetic industry. Many abiotic factors are studied which induce the production of MAAs in algae and cyanobacteria. In this investigation, we have cultivated the green microalga Desmodesmus sp. under salt stress and studied the effect of high salinity on MAAs biosynthesis. MAAs was extracted and partially purified using HPLC method. One compound with similar properties to MAAs was detected from the biomass extract, having a maximum absorption at 320 nm. Accordingly, this genus of microalgae can produce MAA-like compound under this condition, whereas it was not capable to synthesize MAA in normal condition. In fact, salinity is a stressor which can lead to the induction of MAAs synthesis in this microalga. Moreover, this investigation supports that the production of MAAs in microalgae helps the organism to survive in harsh environments, such as high salinity conditions.


HIGHLIGHTS


  • Mycosporine like amino acids (MAAs) can be considered as valuable sunscreens.

  • High salinity condition can induce MAAs production.

  • Desmodesmus sp. in high salinity medium can produce MAA compound.

Purification and Properties of Thermostable Fucoidanase Produced by Recently Isolated Terrestrial Aspergillus flavus FS018

Emmanuel O. Garuba, Paul A. Adeleye, Abiodun A. Onilude

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-7 (e3)
https://doi.org/10.22037/tpps.v5i0.30913

In this study fucoidanase produced by terrestrial Apsergillus flavus FS018 was purified and characterized. The pure fucoidanase enzyme was found to have an optimum activity of 20.8U/mL at 55 ºC and optimum activity of 17.2U/mL at pH 5.0. Furthermore, the fucoidanase retained 96% of its activity after 8 hours of incubation at 55 ºC. Metal ions such Mg2+ and Ca2+ ions were found to slightly enhance the activity of this enzyme while Na+, K+ had inhibitory effect on the activity. The enzyme was found to be active towards fucoidan consisting of α-1→4 and α-1→3 glycoside bonds in the main chains and also galactofucans group. Estimation of the kinetic parameters of the enzyme revealed that Km and Vmax to be 1.9 mM and 0.38 mg/min, respectively when fucoidan from Sargassum vulgare was used as substrate. SDS-PAGE analysis of the purified enzyme revealed that it’s a monomeric enzyme molecule with an estimated molecular weight of 70 kDa.


HIGHLIGHTS


  • Fucoidanase from Aspergillus flavus FS018 was purified and characterized.

  • Molecular weight of the enzyme was estimated to be 70kDa.

  • Enzyme was active towards fucoidan consisting of α-1→4 and α-1→3 glycoside bonds in the main chains and also galactofucans group.

N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase (DapE) a Potential Biomarker of Mycobacterium Tuberculosis: Tag-Free Purification by Metal Affinity Chromatography

Chinmay Umbarkar, Prashant Thakre, Supriya Kashikar, Niraj Ghanwate, Vinod Agarkar

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-9 (e4)
https://doi.org/10.22037/tpps.v5i0.31578

DapE is an enzyme which is highly essential in lysine biosynthetic pathway for the growth and survival of Mycobacterium tuberculosis (Mtb) and other bacterial species although absent in human host. However, the sequence identity of Mtb-DapE with other mycobacterium and bacterial species estimated between 93 to 75% and approximately 50%, respectively. DapE is a metallo-enzyme requires few of transition metals for activity and bacterial proliferation. The homology model based structural studies including published structures revealed an availability of zinc interacting conserved amino residues in Mtb-DapE. In this study, we purified full length recombinant Mtb-DapE as tag-free (Mtb-DapETagFree) protein from inclusion bodies using zinc-NTA column. The single step purified protein observed with 96-98% purity and high yield. The indirect ELISA had 40% sensitivity using Mtb-DapE as an antigen against bovine tuberculosis (bTB) serum samples. IFA analysis with clear fluorescent spots of Mtb native DapE antigen in (3+) human TB positive sputum samples and recombinant Mtb-DapE positive control against Mtb-DapE polyclonal antibody are highly encouraging. The ELISA and IFA results incite for the consideration of Mtb-DapE in future development of quick and ideal TB detection assay along with other mycobacterium antigens. The future advanced detailed structural and functional studies using this highly purified and tag-free Mtb-DapE may provide discovery of antitubercular drug(s) and promising inhibitory molecules.


HIGHLIGHTS


  • Expression and purification of recombinant Mtb-DapETagFree.

  • On-column refolded protein with 98% purity from inclusion bodies purified by zinc-NTA column affinity chromatography.

  • The purified protein observed no aggregation and degradation even after 8 months storage.

  • ELISA and IFA results supports for development of detection assay for TB from tubercular samples.

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Production of Brucella abortus Antiserum in Goats and its Comparison with Conventional Rabbit Antiserum

Sara Mirzaei Samavat, Vahid Kia, Delaram Doroud, Mahdi Paryan, Rahman Shokri

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-7 (e5)
https://doi.org/10.22037/tpps.v5i.32416

Brucellosis, caused by Brucella species, is common among humans and animals, and is one of the most common infectious diseases in Iran. Several assays are available to detect brucellosis, but serological tests may be the only method used in many laboratories. In Iran, different kits are produced in the Pasteur Institute based on agglutination, such as Rose Bengal and 2-mercaptoethanol (2-ME). The positive antiserum control used in these kits is produced in rabbits. The purposeof this study was to produce the antiserum in goats and to compare the titer, quality, and quantity with the antiserum produced in rabbits. The goat immunization was performed by intramuscular injection. Seven days after the last injection, sera were collected. The produced antibody was used in slide and tube agglutination tests with different antigens. The results indicated that the antiserum, produced by the goats had a high quality and quantity. Slide agglutination test showed positive results at 1/6400 dilution with goat antiserum (4+) and 1/1600 dilution with rabbit antiserum (1+). The application of the goats is a better and more appropriate choice, in terms of both cost and quantity, when a high concentration of serum is required. In addition, one goat can provide a higher amount of antiserum compared to several rabbits.


HIGHLIGHTS


  • This study presents a robust and time-saving method for the production of an efficient antiserum.

  • Goat is a better and more appropriate host when higher amount of serum is required.

  • The amount of anti-serum produced in goat is approximately 8 times greater than that of rabbit.

The novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has now been declared as a global pandemic by the World Health Organization (WHO). Several drug molecules have been proposed that can be used against the virus. As an alternative to effective drug molecules, the antiviral peptides have the potential for effective application to control the infectious disease. In this work, the anti- SARS-CoV-2 effect of dermaseptin peptide molecules produced by the skin of the frog was evaluated by using the computational method. Three numbers of antiviral dermaseptin peptides were obtained by searching the antimicrobial peptide database (APD). First, the structure prediction of peptides was done by Pep Fold 2.0 server followed by structure validation by PROCHECK program. Then, the protein-peptide docking simulations were performed using the COVID-19 docking server. The peptides' binding affinity with the SARS-CoV-2 spike protein macromolecule was evaluated along with eight negative control peptides and human angiotensin converting enzymes 2 (ACE2). The protein-peptide docking and interaction analysis resulted in finding that dermaseptin-S9 peptide molecule was the most efficient molecule among the selected peptides with a binding energy of -331.54 KJ/mole. Hence, as a follow-up study, the dermaseptin-S9 peptide molecule can be further designed to enhance its specificity and binding affinity for its better use against the SARSCoV-2 disease.


HIGHLIGHTS


  • Three numbers of antiviral dermaseptin peptides from APD database were in silico evaluated against SARS-CoV-2 spike protein.

  • The antiviral dermaseptin -S9 peptide showed the highest binding affinity towards the SARS-CoV-2 spike protein macromolecule.

  • The hydrophobic property of the distributed amino acids of the derrmaseptin-9 molecule might be related to the binding affinity.

An Investigation into Improvement of Stability and Efficacy of Intravesical BCG Formulations Using Freeze-Drying Technique

Hamidreza Hozouri, Dariush Norouzian, Reza Aboofazeli

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-9 (e7)
https://doi.org/10.22037/tpps.v5i.33393

Bacillus Calmette–Guérin (BCG) has been used as an intravesical product for the treatment of intermediate and high risk, non-muscle-invasive bladder cancer (NMIBC). Freeze drying technique is highly recommended for product development, however, the microorganism sensitivity to freezing and drying processes is a major chalenge which may lead to poor survival. To overcome this problem, the use of cryoprotectants in intravesical BCG formulation is required. This study was, therefre, planned to design a new formulation, using an attenuated strain of Mycobacterium bovis, which could be produced by freeze drying technique with the aim of prolonging its storage stability and increasing its efficacy as well as the ease of administration. For this purpose, sodium L-glutamate monohydrate (a commonly used stabilizer in domestic BCG suspension formulations) was replaced by lactose monohydrate. New intravesical BCG formulations, both in lyophilized and liquid forms, were eventually evaluated by moisture content assay, viable count assay, bacterial and fungal contamination, safety test and determination of bacterial concentration and O2 consumption. The results were compared with the data obtained for the conventional lyophilized and liquid products. Maximum survival rate was achieved in the presence of 10 % w/v lactose monohydrate for both liquid and lyophilized formulations when stored at less than -10 and 2-8C, respectively. In summary, the freeze-dried formulations developed with lactose monohydrate met the requirements of intravesical BCG in high viability and stability during storage.


HIGHLIGHTS


  • Lyophilization technique and lactose monohydrate as a protecting sugar were used to improve the stability, storage conditions, shelf-life and effcicay of intravesical BCG product.

  • Maximum survival rate was attained for both liquid and lyophilized lactose-based formulations prepared with 10 %w/v lactose monohydrate and stored at less than -10 and 2-8°C, respectively.

  • Freeze-dried products possessed higher stability and efficacy in comparison with corresponding liquid preparations.

 

Infrared Free Electron Laser or Polarized Ultraviolet Photolysis of Hierarchical and Chiral Components of Interleukin-6, AlanylAlanine and Alanine

Nanami Fujisawa, Yuika Onami, Takayasu Kawasaki, Tomoyuki Haraguchi, Takashiro Akitsu, Koichi Tsukiyama

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-11 (e8)
https://doi.org/10.22037/tpps.v5i.33129

Interleukin-6 (IL-6) could be decomposed by irradiation of IR-FEL (Infrared free electron laser). Using circularly polarized and other UV light and IR-FEL light, photolysis of hierarchical components of cast films of IL-6, namely deuterated aqueous solutions of enantiomers of dipeptide (L-alanyl-L-alanine (Ala-ala) or D-alanyl-D-alanine) and enantiomers of amino acid (L-alanine (Ala) or D-alanine) was investigated whether specific bonds can be broken by absorption of light (not due to heat). In addition, IR-FEL irradiation to powder as well as crystal structure determination for L-Ala and D-Ala at 173 and 293 K were also carried out to confirm reproducibility in the solid state about long-lasting controversy about Salam’ hypothesis associated with chirality exhibiting structural phase transition at different temperature. Subunits of IL-6 (dipeptide and amino acid) could not be decomposed by polarized IR-FEL nor UV (ultraviolet) light regardless of their chirality. All experimental methods tested in this study failed to prove Salam's hypothesis, positively. Consequently, secondary structure of IL-6 was found to be easier to be damaged by IR-FEL than covalent bonds.


HIGHLIGHTS


  • Interleukin 6 (IL-6) was decomposed by irradiation of IR-FEL (Infrared free electron laser).

  • Parts of IL-6 (dipeptide and amino acid) was not decomposed by polarized IR-FEL nor UV (ultraviolet) light regardless of their chirality.

  • Secondary structure of IL-6 was easier to be damaged by IR-FEL than covalent bonds.

 

A Rapid and Cost-Effective Method for the Purification of Streptococcus pneumoniae Antiserum Using Tangential Flow Filtration Method

Mitra Moghiman, Vahid Kia, Zahra Panahi, Rahman Shokri; Mahdi paryan

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-6 (e9)
https://doi.org/10.22037/tpps.v5i.33390

Antibodies are important agents in the laboratory diagnosis of various microorganisms such as Streptococcus pneumoniae. In this study, we prepared and purified IgG antibody against Streptococcus pneumoniae using novel methods to be used in ELISA and agglutination diagnostic kits. First, Streptococcus pneumoniae was cultured, harvested, and inactivated. Then, bacteria were injected into four mature New Zealand white rabbits, and antisera were developed. Afterward, immunoglobulins were purified using ammonium sulfate precipitation, diafilteration using Tangential Flow Filtration, and ion-exchange chromatography. The purified antibody was then biotinylated and used in ELISA. Gel electrophoresis results showed that the antibody was highly pure. Agglutination test on the primary antigen and clinical samples was four-plus. ELISA results showed that the sensitivity and specificity of the test were 95% and 100%, respectively. Results indicated that our fast method was suitable for anti-Streptococcus pneumoniae IgG purification. Repetitive qualitative and quantitative experiments confirmed high purity of the immunoglobulin. Thus, it could be a suitable candidate to be used in laboratory diagnostic kits.


HIGHLIGHTS


  • A rapid and cost-effective method for the purification of Streptococcus pneumoniae antiserum.

  • IgG antibody was prepared and purified by using tangential flow filtration method.

  • The purified antibody can be used in ELISA and agglutination diagnostic kits.

Author Package, TPPS, Vol. 5 (2020)

Maryam Tabarzad

Trends in Peptide and Protein Sciences, Vol. 5 (2020), , Page 1-12
https://doi.org/10.22037/tpps.v5i.37265

The Trends in Peptide and Protein Sciences is a peer-reviewed, online-only (previously print-online), scientific journal owned by Protein Technology Research Center, Shahid Beheshti University of Medical Sciences and documents in all important aspects of the research in peptides and proteins focusing on analytics and impurities, bioinformatics, biopharmaceuticals and vaccines, biotechnology, chemical synthesis, conformational analysis, design and  development of protein therapeutics, determination of structure, enzymology, folding and sequencing,  formulation and stability, function, genetics,  immunology, kinetics, modeling, molecular biology, pharmacokinetics and pharmacodynamics of therapeutic proteins and antibodies, pharmacology,  protein engineering and development, protein-protein interaction, proteomics, purification/expression/production, simulation, thermodynamics and  hydrodynamics and protein biomarkers. The aim of this Journal is to publish high quality original research articles, reviews, short communications and letters and to provide a medium for scientists and researchers to share their findings from the area of peptides and proteins. The Trends in Peptide and Protein Sciences is published in collaboration with Iranian Association of Pharmaceutical Scientists. From volume 3 (2018) of TPPS, articles are continuously published online only, as soon as the review process is completed.