Evaluation of Activity Kinetic Parameters of SK319cys, As a New Cysteine Variant of Streptokinase: A Comparative Study

Background:Despite the extensive use of streptokinase in thrombolytic therapy, its administration may have some shortcomings like allergic reactions and relatively low half life. Specific PEGylation on cysteine at desired sites of streptokinase may alleviate these deficiencies and improve the quality of treatment.Objective:This study was carried out to create a new cystein variant of streptokinase and compare its activity with formerly mutated SK263cys, SK45cys and intact streptokinase (Ski) to introduce superior candidates for specific PEGylation.Method:In silico study was carried out to select appropriate amino acid for cysteine substitution and accordingly mutagenesis was carried out by SOEing PCR. The mutated gene was cloned in E. coli, expressed, and purified by affinity chromatography. Activity of the purified proteins was assayed and kinetic parameters of enzymatic reaction were analyzed.Results:According to in silico data, Arginine319 was selected for substitution with cysteine. SK319cys was achieved with 98% purity after cloning, expression and purification. It was shown that the enzymatic efficiency of SK319Cys and SK263cys was increased 18 and 21%, respectively, when compared to SKi (79.4 and 81.3 vs. 67.1µM-1min-1), while SK45cys showed 7% activity decrease (62.47µM-1min-1) compared to SKi. According to time-based activity assay, SK319Cys and SK263cys exhibited higher activity at lower substrate concentrations (100 and 200 µM), but at higher concentrations of substrate (400 and 800 µM), the proteins showed a very close trend of activity.Conclusion:SK319cys, as the new cysteine variant of streptokinase, together with SK263cys and SK45cys can be considered as appropriate molecules for specific PEGylation.

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An in silico structural insights into Plasmodium LytB protein and its inhibition

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314082096 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1791-C1791

Author(s):

Rajabrata Bhunya ◽

Suman Nandy ◽

Alpana Seal

Keyword(s):

In Silico ◽

3D Structure ◽

Three Dimensional ◽

Comparative Modeling ◽

Binding Energies ◽

E Coli ◽

Dimethylallyl Diphosphate ◽

In Silico Study ◽

Structural Insights ◽

Insight Into

In most of the pathogenic organisms including Plasmodium falciparum, isoprenoids are synthesized via MEP (MethylErythritol 4-Phosphate) pathway. LytB is the last enzyme of this pathway which catalyzes the conversion of (E)-4-hydroxy-3-methylbut-2-en-1-yl diphosphate (HMBPP) into the two isoprenoid precursors: isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Since the MEP pathway is not used by humans, it represents an attractive target for the development of new antimalarial compounds or inhibitors. Here a systematic in-silico study has been conducted to get an insight into the structure of Plasmodium lytB as well as its affinities towards different inhibitors. We used comparative modeling technique to predict the three dimensional (3D) structure of Plasmodium LytB taking E. Coli LytB protein (PDB ID: 3KE8) as template and the model was subsequently refined through molecular dynamics (MD) simulation. A large ligand dataset containing diphospate group was subjected for virtual screening against the target using GOLD 5.2 program. Considering the mode of binding and affinities, 17 leads were selected on basis of binding energies in comparison to its substrate HMBPP (Gold.Chemscore.DG: -20.9734 kcal/mol). Among them, 5 were discarded because of their inhibitory activity towards other human enzymes. The rest 12 potential leads carry all the properties of any "drug like" molecule and the knowledge of Plasmodium LytB inhibitory mechanism which can provide valuable support for the antimalarial inhibitor design in future.

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The Comparison Between the Mutated HuIFN-β 27-101 and the WildType Interferon β: the Comprehensive In Silico Study to Evaluate theEffect of Mutations on IFN-β

Advanced Pharmaceutical Bulletin ◽

10.15171/apb.2019.074 ◽

2019 ◽

Vol 9 (4) ◽

pp. 640-648

Author(s):

Sayed Sharif Balkhi ◽

Zohreh Hojati

Keyword(s):

Molecular Docking ◽

In Silico ◽

Interferon Beta ◽

Human Interferon ◽

Meaningful Relationship ◽

Significant Difference ◽

In Silico Study ◽

The Impact ◽

Life Function

Purpose: Interferon beta (IFN-β) is used to combat multiple sclerosis (MS) disease. CreatingR27T and V101F mutations (mHuIFN-β-27 and mHuIFN-β-101) is one of the tasks performedto improve human interferon beta (HuIFN-β) half-life, function and expression. In this work,the impact of R27T and V101F mutations in recombinant IFN-β on its binding to interferonreceptors were studied by molecular docking.Methods: This work was performed through in silico study. The simulation of mutation wasperformed using the online Rosetta Backrub software and checked using server verify3D.Comparison of access to the solvent of the amino acids in the structures created was performedusing the asaview online server. Also, the effect of mutations on the fold of the protein wasreviewed by the online HOPE server. The molecular docking was performed between HuIFN-βand the external region of IFNAR receptor using the online ClusPro2 protein-protein dockingserver.Results: The comparison of the values of the negative binding energy (ΔGbind) obtained fromprotein-protein molecular docking between IFNAR receptor and HuIFN-β, mHuIFN-β-27,mHuIFN-β-101 and mHuIFN-β-27-101 ligands did not show a significant difference, and thesedifferences do not see any meaningful relationship between them (P > 0.9999).Conclusion: Regarding these results, it can be concluded that these mutations do not have anegative effect on the composition of the complex rHuIFN-β/IFNAR. So, they do not interferewith the binding of the IFN-β to the receptor. It is concluded that the quality of the rHuIFN-β isimproved by introducing these two mutations.<br />

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Increasing thermal stability and catalytic activity of glutamate decarboxylase in E. coli: An in silico study

Computational Biology and Chemistry ◽

10.1016/j.compbiolchem.2016.05.006 ◽

2016 ◽

Vol 64 ◽

pp. 74-81 ◽

Cited By ~ 4

Author(s):

Yasaman Tavakoli ◽

Abolghasem Esmaeili ◽

Hossein Saber

Keyword(s):

Thermal Stability ◽

Catalytic Activity ◽

In Silico ◽

Glutamate Decarboxylase ◽

E Coli ◽

In Silico Study

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THE INFLUENCE OF ETHANOL ON THE CONVERSION OF PREGNENOLONE TO PROGESTERONE BY HUMAN PLACENTAL MICROSOMES

Acta Endocrinologica ◽

10.1530/acta.0.0760178 ◽

1974 ◽

Vol 76 (1) ◽

pp. 178-188 ◽

Cited By ~ 3

Author(s):

H. Lübbert ◽

K. Pollow ◽

R. Wagner ◽

J. Hammerstein

Keyword(s):

Kinetic Parameters ◽

Enzyme Preparation ◽

Ethanol Concentration ◽

Hydroxysteroid Dehydrogenase ◽

Product Formation ◽

Linear Increase ◽

Maximal Velocity ◽

Microsomal Enzyme ◽

Temperature Optima ◽

Substrate Concentrations

ABSTRACT The effects of ethanol on kinetic parameters of placental Δ5-3β-hydroxysteroid dehydrogenase were studied. In the presence of high pregnenolone concentrations (50 μm, [S] > Km) the microsomal enzyme preparation exhibited an almost linear increase in activity as the ethanol concentration in the medium was raised from 2.5 to 15 % (v/v). At lower substrate concentrations ([S] << Km) ethanol caused inhibition. Other effects of ethanol were: linearity of product formation with time was prolonged; the maximal velocity was markedly increased; the Km for pregnenolone slightly decreased with increasing ethanol concentrations (2.5 to 10 %, v/v) whereas the Km for NAD remained the same. The pH and temperature optima of the reaction were unaffected by ethanol. Other organic solvents caused similar effects.

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STUDI PERBANDINGAN KUALITAS AIR BERSIH DALAM KAITANYA DENGAN AKTIVITAS MASYARAKAT DI SEKITAR SUMBER AIR PADA DESA WAINITU, BATUMERAH, AMAHUSU, DAN HALONG

JURNAL BUDIDAYA PERTANIAN ◽

10.30598/jbdp.2017.13.2.111 ◽

2017 ◽

Vol 13 (2) ◽

pp. 111-119

Author(s):

Lela Uyara ◽

Pieter Kunu ◽

Silwanus M Talakua

Keyword(s):

Water Quality ◽

Quality Standard ◽

Total Coliform ◽

Quality Analysis ◽

Clean Water ◽

Water Quality Analysis ◽

E Coli ◽

Microbiological Parameters ◽

Standard Quality

The study aims to determine the quality of clean water in the villages of Wainitu, Batumerah, Amahusu and Halong by comparing the result of water quality analysis with water quality standard. Water quality analysis includes Physiscal, Chemical, and Microbiological parameters. This research uses descriptive method, this method describes systematics, accurate about facts and characteristic of the quality of clean water of each research location. The results showed that the source of clean water in the village of Batumerah did not meet the standard of clean water quality standards indicated by the number of E. coli and the high total coliform. Keywords: standard quality of clean water, water quality, Wainitu, Batumerah, Amahusu and Halong villages ABSTRAK Penelitian yang bertujuan untuk menetapkan kualitas air bersih di Desa Wainitu, Batumerah, Amahusu dan Halong, dengan membandingkan hasil analisis kualitas air dengan standar baku mutu air bersih. Analisis kualitas air meliputi parameter fisika, kimia dan mikrobiologi. Penelitian ini menggunakan metode deskriptif; metode ini menggambarkan sicara sistematis, akurat, fakta dan karakteristik mengenai kualitas air bersih di masing-masing lokasi penelitian. Hasil penelitian menunjukkan bahwa sumber air bersih di Desa Batumerah tidak memenuhi standar baku mutu air bersih yang ditunjukkan oleh jumlah E. coli dan total Koliform yang tinggi. Kata Kunci: baku mutu air bersih, Desa Wainitu, Batumerah, Amahusu dan Halong, kualitas air

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The Effect of Glargine As Basal Insulin Support for Recovering Critically Ill and High Dependency Unit Patients: An in Silico Study

7th IFAC Symposium on Modelling and Control in Biomedical Systems ◽

10.3182/20090812-3-dk-2006.00009 ◽

2009 ◽

Author(s):

Lin, Jessica

Keyword(s):

Critically Ill ◽

In Silico ◽

Basal Insulin ◽

High Dependency Unit ◽

High Dependency ◽

In Silico Study

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Impact of Raw Materials and Processing Techniques on The Microbiological Quality of Egyptian Domiati Cheese

International Journal of Veterinary Science ◽

10.37422/ijvs/20.060 ◽

2020 ◽

Keyword(s):

Raw Materials ◽

Microbiological Quality ◽

Raw Milk ◽

Plate Count ◽

E Coli ◽

Fresh Cheese ◽

Processing Techniques ◽

The Impact ◽

Total Yeast

Domiati cheese is the most popular brand of cheese ripened in brine in the Middle East in terms of consumed quantities. This study was performed to investigate the impact of the microbiological quality of the used raw materials, the applied traditional processing techniques and ripening period on the quality and safety of the produced cheese. Three hundred random composite samples were collected from three factories at Fayoum Governorate, Egypt. Collected samples represent twenty-five each of: raw milk, table salt, calf rennet, microbial rennet, water, environmental air, whey, fresh cheese, ripened cheese & swabs from: worker hands; cheese molds and utensils; tanks. All samples were examined microbiologically for Standard Plate Count (SPC), coliforms count, Staphylococcus aureus (S. aureus) count, total yeast & mould count, presence of E. coli, Salmonellae and Listeria monocytogenes (L. monocytogenes). The mean value of SPC, coliforms, S. aureus and total yeast & mould counts ranged from (79×102 CFU/m3 for air to 13×108 CFU/g for fresh cheese), (7×102 MPN/ cm2 for tank swabs to 80×106 MPN/ml for raw milk), (9×102 CFU/g for salt to 69×106 CFU/g for fresh cheese) and (2×102 CFU/cm2 for hand swabs to 60×104 CFU/g for fresh cheese), respectively. Whereas, E. coli, Salmonella and L. monocytogenes failed to be detected in all examined samples. There were significant differences in all determined microbiological parameters (p ≤0.05) between fresh and ripened cheese which may be attributed to different adverse conditions such as water activity, pH, salt content and temperature carried out to improve the quality of the product.

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Water Quality of Swimming Pools in Jerusalem

Water Science & Technology ◽

10.2166/wst.1993.0562 ◽

1993 ◽

Vol 27 (7-8) ◽

pp. 287-294 ◽

Cited By ~ 2

Author(s):

S. Lerman ◽

O. Lev ◽

A. Adin ◽

E. Katzenelson

Keyword(s):

Water Quality ◽

Fecal Coliforms ◽

Pathogenic Microorganisms ◽

Swimming Pools ◽

Indicator Organisms ◽

Indicator Microorganisms ◽

E Coli ◽

Safe Water ◽

Indirect Information

The Israel Ministry of Health is now revising its regulations for the assurance of safe water quality in public swimming pools. Since it is not possible to monitor each of the pathogenic microorganisms, it is often recommended to monitor indicator bacteria which provide indirect information on the water quality in the swimming pool. Three indicator microorganisms are often recommended: coliform counts (total coliforms, fecal coliforms or E. Coli), staphylococcus aureus and pseudomonas aeruginosa. A four year survey of the water quality of swimming pools in the Jerusalem District was conducted in order to determine whether the monitoring of all three indicators is necessary to assure safe water quality or is it sufficient to monitor only a single microorganism. A statistical analysis, conducted by using several different statistical techniques, reveals that the populations of the three indicator organisms are significantly interdependent but the correlations between each pair of these indicators are not sufficient to base a prediction of any of the organisms based on the measurements of the others. Therefore, it is concluded that monitoring of all three indicators should be recommended in order to provide an adequate picture of the water quality in swimming pools.

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Production and Preliminary In Vivo Evaluations of a Novel in silico-designed L2-based Potential HPV Vaccine

Current Pharmaceutical Biotechnology ◽

10.2174/1389201020666191114104850 ◽

2020 ◽

Vol 21 (4) ◽

pp. 316-324

Author(s):

Manica Negahdaripour ◽

Navid Nezafat ◽

Reza Heidari ◽

Nasrollah Erfani ◽

Nasim Hajighahramani ◽

...

Keyword(s):

In Silico ◽

Group Versus ◽

Tlr Agonists ◽

E Coli ◽

Metal Affinity ◽

Th1 And Th2 Cytokines ◽

Ifn Γ ◽

Humoral Responses ◽

Future Work

Background: L2-based Human Papillomavirus (HPV) prophylactic vaccines, containing epitopes from HPV minor capsid proteins, are under investigation as second-generation HPV vaccines. No such vaccine has passed clinical trials yet, mainly due to the low immunogenicity of peptide vaccines; so efforts are being continued. A candidate vaccine composed of two HPV16 L2 epitopes, flagellin and a Toll-Like Receptor (TLR) 4 agonist (RS09) as adjuvants, and two universal T-helper epitopes was designed in silico in our previous researches. Methods: The designed vaccine construct was expressed in E. coli BL21 (DE3) and purified through metal affinity chromatography. Following mice vaccination, blood samples underwent ELISA and flow cytometry analyses for the detection of IgG and seven Th1 and Th2 cytokines. Results: Following immunization, Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-5, IL-10) type cytokines, as well as IgG, were induced significantly compared with the PBS group. Significant increases in IFN-γ, IL-2, and IL-5 levels were observed in the vaccinated group versus Freund’s adjuvant group. Conclusion: The obtained cytokine induction profile implied both cellular and humoral responses, with a more Th-1 favored trend. However, an analysis of specific antibodies against L2 is required to confirm humoral responses. No significant elevation in inflammatory cytokines, (IL-6 and TNF-α), suggested a lack of unwanted inflammatory side effects despite using a combination of two TLR agonists. The designed construct might be capable of inducing adaptive and innate immunity; nevertheless, comprehensive immune tests were not conducted at this stage and will be a matter of future work.

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