International Journal of Current Research and Review
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IJCRR - Vol 03 Issue 07, July, 2011

Pages: 04-14

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ISOLATION AND CHARACTERIZATION OF LOLDUVIN-S: A NOVEL ANTIMICROBIAL PROTEIN FROM THE INK OF INDIANS QUID LOLIGO DUVAUCELI

Author: Smiline Girija, J.Vijayshree Priyadharshini, Pandi Suba K., Hariprasad G., Raghuraman R.

Category: General Sciences

Abstract:Objectives: Bioactive compounds from the marine habitat have been always represented as the greatest unexploited source of potentially active pharmaceutical agents. Squid ink, being reported for its various therapeutic applications has not been extensively studied for its bioactive molecules. Thus an attempt has been made in this study to isolate and characterize a novel antimicrobial protein from the ink of Loligo duvauceli. Methods: The fresh squids were decapitated and the ink sac was dissected to collect the ink. Melanin free ink was obtained by ultracentrifugation. The ink was then subjected to SDSPAGE to analyze the proteins present in it and a new protein with a molecular weight of approximately 60 Kda was selected as the target protein. Native PAGE was then performed to isolate the protein in an original form and was eluted using passive gel elution method. The eluted protein was then subjected to an enzyme linked coupled assay for the L-amino oxidase [LAO] activity. The Km and Vmax values for the L-amino acids utilized as substrates for the assay were determined by Lineweaver Burk Plots. The eluted protein was sterilized by syringe filtration after which the antimicrobial potential of the protein was checked by agar well diffusion method against the drug resistant pathogens such as ESBL, MRSA and the pathogenic yeast C.albicans. The MIC value of the protein was determined by Microbroth dilution method.
Results:
A new protein of approximately 60 Kda was successfully isolated and was characterized to exhibit the L-amino acid oxidase activity. The protein was scored to possess a promising antibacterial and antifungal activity against the test pathogens. The MIC value was determined as 25 \?g/ml for ESBL producing strains of E.coli and K.pneumoniae. MIC value for the methicillin resistant S.aureus and C.albicans was deduced as 12.5 \?g/ml. This new protein exhibiting a LAO activity was further named as Lolduvin - S. Conclusion: This study was concluded by stating that Lolduvin-S exhibits the LAO activity and had been reported to possess a promising antimicrobial activity against the dreadful drug resistant pathogens. Lolduvin-S could be employed in near future as a novel therapeutic agent to treat various systemic ailments.

Keywords: Antimicrobial, drug resistant pathogens, Microbroth dilution method.

Full Text:

INTRODUCTION

Natural bioactive substances have the least quantum of side effects when compared with the synthetic products. Although most antibiotics were derived from terrestrial life, it is the marine world that may provide the pharmaceutical industry with the next generation of medicines. At present, a number of marine natural products are in the market or in clinical trials. Due to the huge diversity of marine bioactive compounds with respect to their chemical structure, mode of action and applicability, the extreme concern has been increased towards the screening of marine natural products for their biomedical potential1 . In spite of incredible evolution in medicinal field, some deadly diseases are not curable. The emergence and spread of antimicrobial resistance is now threatening to undermine our ability to treat infections and save lives. Drug resistant bacteria such as extended spectrum beta lactamase (ESBL) producing strains of Escherichia coli, Klebsiella pneumoniae, methicillin resistant Staphylococcus aureus (MRSA) continue to cause a large number of infections and deaths particularly in developing countries2 . Thus a novel therapeutic compound from an alternate source with unique metabolic and physiological capabilities producing different kinds of metabolites must be employed for future therapeutic scenario globally. Squids under cephalopod family, is known to produce a black pigmented ink as a defensive ploy against its predators. This ink has already been reported to exhibit various therapeutic values3 . The crude ink extracts from various species of cephalopods have already been studied for its antimicrobial potential against biofilm bacteria, Staphylococcus aureus4 , clinical bacterial isolates and pathogenic yeasts5 , preservative property6 , antioxidant values7 and anti-retroviral activity8 . The ink has also been studied for its various bioactive proteins possessing cytolytic property9 , antitumour fractions10 and antimicrobial potential11. With these review background this study has been aimed at isolating and characterizing a new and novel protein from the ink of the Indian squid Loligo duvauceli and to study its antimicrobial potential against the drug resistant pathogens such as MRSA and ESBL producing strains of E.coli and K.pneumoniae.

METHODS

Collection and dissection of squids: Fresh squids were caught from the south west coast of Chennai and were decapitated by the fishermen. The squid was fixed in 4% formalin (4ml formalin in 96ml of water) and was identified as Loligo duvauceli by an eminent Zoologist based on the Zoological taxonomy. The dissection of the squids was performed as per the instructions of the Zoologist. After dissection of the ink sac the surface of the gland was sterilized with ethanol and was blotted with sterile cotton. The ink duct was cut with sterile scissors and the sac was gently squeezed and the excreted ink was collected in sterile brown bottles. The ink was stored at 4ºC until use.

Preparation of melanin free ink: The extracted fresh ink was subjected to centrifugation under cold condition at 15,000 rpm for 30 min to remove melanin. The supernatant was used as melanin free ink. The samples were frozen and kept at - 80°C until use.

Estimation of protein

The protein estimation was done by Lowry‘s method12 , a protein assay that is based on the reaction where the peptide bonds of proteins react with copper under alkaline conditions producing Cu+ ions that react with the Folin‘s reagent producing blue colour.

SDS Polyacrylamide gel electrophoresis:

SDS-PAGE was carried out using an acrylamide concentration of 7.5% in the resolving gel13. Stock solutions were prepared as recommended14 and stored in brown bottles at 4?C. The separating gel mix (12%) was prepared in a 25ml conical flask using acrylamide monomer 2.8ml, separating gel buffer (4x) 1.750 ml, 10 % SDS 0.070 ml, 10% APS 0.035 μl and distilled water 2.345 ml (Final volume – 7ml). The solution was de-gased for 5 min and 3 μl of TEMED was added with gentle mixing and was poured into the glass plates. 100 μl of N-butanol was added over the gel mix and the arrangement was left undisturbed for 20 min. The stacking gel mix (5%) was prepared by mixing acrylamide monomer 0.333 ml, 4x Stacking Gel Buffer 0.500 ml, 10 % SDS 0.020 ml, 10% APS 0.010 μl and distilled water 1.137 ml (Final volume – 2.0 ml). The electrophoretic run was performed as per standard protocols by adding the samples to the wells along with a molecular weight marker. Staining was done using Coomasie brilliant blue and destaining was done with ethanol and acetic acid. The gel was then analysed in the documentation unit. The molecular weight of the unknown peptides was thus determined by comparing it with the standard molecular weight marker bands.

Native Polyacrylamide gel electrophoresis:

Native – PAGE was performed without SDS using the same methodology as described above. Staining was not done. The unstained gel was then carefully removed and the target protein band portion corresponding to target protein band size was excised in sterile phosphate buffer and was eluted as follows.

Passive gel elution of the target protein:

Elution of the protein was performed by passively eluting the proteins from polyacrylamide gel pieces15. The excised gel pieces corresponding to the target protein band were placed in a clean microcentrifuge tubes. The elution buffer was prepared using Tris HCl 50 mM, NaCl 150 mM and EDTA 0.1 mM (pH 7.5). 0.5- 1ml of cold elution buffer is added so that the gel pieces were completely immersed. The gel pieces were crushed using a sterile scalpel and was incubated in a rotary shaker at 30ºC overnight. Centrifugation was done at 5000-10000g for 10 minutes and the supernatant was carefully pipetted into a new microcentrifuge tube. An aliquot of the supernatant might be tested for the presence of the same protein by subjecting it to SDS-PAGE

Enzymatic assay for L – amino acid oxidase [LAO] activity:

LAO activity of the protein was performed by an enzyme coupled assay16. Purified protein was prepared for the assay in 50 mM/L phosphate buffer and 150 mM/L KCl. The reaction mixture was prepared as follows: Tris HCl 0.1 mM/L (pH 7.6), Horseradish peroxide 10 μg, O-dianisidine 0.2 mM/L and L-amino acids 2 mM/L. The test sample was mixed with 100 μl of the reaction mixture. Reaction was performed at room temperature for 1-60 min. The activity was monitored by absorbance at 436 nm. The increase in absorbance was transformed into molar concentration of the end product o-dianisidine (8.31 x 103 mol/L). The Km and Vmax values of the Lamino acids were determined by Lineweaver-Burk plots.

Antimicrobial bioassay:

The antimicrobial assay was performed by Nathan‘s agar well diffusion method17 with the drug resistant strains that included the extended spectrum beta lactamase (ESBL) producing strains of E.coli and K.pneumoniae, methicillin resistant S.aureus (MRSA) and Amphotericin B resistant C.albicans isolated from various clinical specimens from the patients visiting the outpatient unit of Meenakshi General Hospital (Data not given). The control strains include E.coli (ATCC 25922), K.pneumoniae (ATCC 10031), S.aureus (ATCC 25923) and C.albicans (ATCC 10231).

A minimum of four colonies of the test organisms were touched with a sterile loop and transferred into Sterile Mueller Hinton broth and C.albicans into Saboraud‘s dextrose broth under aseptic conditions and was incubated for two hours at 37ºC. After incubation the density of each microbial suspension was adjusted equal to that of 106 c.f.u/ml (standardized by 0.5 MacFarland standards) and was used as the inoculum.

Agar well diffusion assay

Fifty microlitres (50μl) of inoculum of each test and control organism was spread as lawn cultures onto sterile Mueller Hinton agar plates using L-rods to achieve a confluent growth. The agar plates were allowed to dry and wells or cups of 8 mm were made with a sterile agar borer in the inoculated agar plates. The eluted protein was subjected to syringe membrane filtration prior to the bioassay and an aliquot of the same was streaked onto nutrient agar plates and was incubated at 37 C for 24 hrs for sterility checking. A 50 μl volume of the eluted protein in phosphate buffered saline was propelled directly into the wells of the inoculated specific media agar plates for each test organism. The plates were then allowed to stand for 10 minutes for diffusion of the protein to take place and incubated at 37ºC for 24 hrs. Sterile DMSO served as Negative control. Erythromycin 30 µg (for bacteria) and Amphotericin B 100 U (for yeast) were included as positive controls. After incubation the plates were observed for the zone of inhibition around the wells and the zone size was measured using an antibiotic sensitivity measuring scale (Himedia). The antimicrobial efficacy was graded based on the zone diameter as high activity (> 15 mm), moderately active (10- 14 mm), trace activity (5-9 mm) and no activity (< 4 mm)18 . Determination of MIC value for the protein MIC value for the purified protein was determined by Microbroth dilution method19. Serial dilutions of the eluted protein were done in a 96 well microtitre plate with sterile phosphate buffer. The dilution factor was 5, 2.5, 1.25, 0.625, 0.312 and 0.156 μg/100 μl. To each dilution 100 μl of the culture broths of the test strains and control strains were added in their respective wells and the plate was incubated at 37 C for 24 hrs. After incubation the spectrophotometric analysis was performed and the OD values were recorded. The MIC value was also confirmed with Microbial Spot Checker board method20 where 3 μl of each dilution was spotted onto Mueller Hinton agar plates and incubated at 37 C for 24 hrs. After incubation the spot showing the complete absence of microbial growth indicates the minimum inhibitory concentration value.

RESULTS

Centrifugation of the crude ink at 15,000 rpm for 15 min yielded melanin free ink. Using Lowry‘s method, a standard curve of absorbance was obtained as a function of initial protein concentration and it was used to determine the unknown protein concentration. Thus the total protein in the melanin free ink was determined as 65.5 mg/ml. SDS PAGE showed a major protein band with a molecular weight of 60 KDa. Two weak bands were also obtained and their molecular weights were determined as 100 KDa and 80 KDa (Figure 1). The 60 Kda band was the target protein of the study as it has not been reported earlier. The protein was successfully isolated by Native Gel Electrophoresis (Figure 2) and was eluted by passive gel elution technique. L-amino acid oxidase (LAO) assay performed showed that when the purified protein and 2 mM/L amino acids were incubated for 1 min, L-lysine and LArginine proved to serve as excellent LAO substrates. Km and Vmax values of lysine were 33-87 µmol and 1.93-2.71 µmol/sec respectively and Km and Vmax values for arginine is 25-125 µmol and 1.56-3.30 µmol/sec. The reactions were completed within 30 secs at room temperature for lysine and arginine at a concentration of 0.02-2 mM/L. Thus in this study we report a novel protein with LAO activity from the ink of Loligo duvauceli which has been named as Lolduvin-S. The protein after membrane filtration yielded no growth on nutrient agar plate; thus it was sterile and was used for bioactivity assays. Lolduvin-S had scored to possess high antibacterial activity against the drug resistant test strains (Table 1). The zone size ranges from a mean value of 20 mm for E.coli (ESBL), 21 mm for K.pneumoniae (ESBL), 22 mm for S.aureus (MRSA) and 20 mm for C.albicans. The zone size was compared with the control strains (Figure 3). The MIC value was determined as 25 µg/ml for E.coli and K.pneumoniae, 12.5 µg/ml for S.aureus and C.albicans. The previous dilution that showed the visible decrease in the number of colonies was determined as the bacteriostatic dose and was deduced as 12.5 µg/ml for E.coli and K.pneumoniae, 6.25 µg/ml for S.aureus and C.albicans. Thus a novel protein called Lolduvin-S has been isolated and characterized to possess a potent LAO activity from the ink of Loligo duvauceli. The protein had also a promising antimicrobial activity against the dreadful resistant pathogens like ESBL and MRSA organisms.

DISCUSSION

The marine ecosystem offers rich niche for marine flora and fauna. The protein structures, metabolic pathways, reproductive systems, sensory and defense mechanisms developed by marine microorganisms and other lifeforms are distinctive in nature. During the last few decades several novel compounds in point of view of potential drug development have been isolated from marine habitat with various therapeutic applications21. The antimicrobial property of the squid ink is already a known fact and the ink has been found to posses a promising antimicrobial effect in our previous studies. The proteins present in various species of gastropods and cephalopods have been characterized earlier22. This study is thus undertaken to characterize a novel antimicrobial protein from the ink of Loligo duvauceli. The melanin free ink has been used in various studies for the analysis of the protein and it holds suitable for various protein analysis The total protein estimated in this study from the squid ink suggests the availability of the bioactive protein for various pharmaceutical applications. Determination of the molecular weight of the proteins is best achieved by the bioinstrumentational tool such as the SDS PAGE. The electrophoretic separation of the proteins from other species of squid has been achieved with 2.5% and 7.5% gel resolution23. In this study we employed 12.5% gel that is suitable for the separation of the protein from Loligo duvauceli‘s ink. Apart from the major protein band at 60 Kda, two other weak bands at 100 Kda and 80 Kda have also been observed. This shows the presence of tyrosinase24 and peroxidase25 respectively which has been already reported from the ink of various cephalopods and squids. The 60 Kda protein in Loligo duvauceli has not been reported anywhere earlier and thus it has been considered as the target protein. Native PAGE separates the protein in a pure form without denaturation though effective and high amount of proteins can be obtained with gel filtration chromatography methods26. Due to high cost, gel filtration chromatography is not done for the isolation of the proteins: thus the passive gel elution protocol suggested by the Pierce technologies, USA is followed for the same. It is a simple and rapid protocol for the elution of target protein. The elution protocol is slightly modified by performing the elution under cold condition. The protein eluted is rechecked by subjecting the protein for SDS-PAGE and by obtaining the same band at 60 Kda. A similar type of protein called Escapin with a molecular weight of 60 Kda has been reported from the ink of a sea hare under the Phylum Mollusca earlier27 . Escapin is known for its L-amino oxidase activity which is a hydrogen peroxide induced destruction of the target cells (such as micro-organisms) in the presence of Lamino acids. With this view an attempt to check the LAO activity of Lolduvin-S protein is done by Enzyme linked coupled assay. Lolduvin-S has been found to possess an enzymatic L-amino acid oxidase activity. Hydrogen peroxide is sufficient to cause bacteriostasis and does so with a concentration dependency and threshold similar to that of amino acids as substrates for the enzymatic activity. Both lysine and arginine are substrates for LAO activity from the squid ink. It shows strong and rapid activity in this study when using arginine or lysine as substrates, with reactions being completed within 30 secs at room temperature. A similar protein has been reported to be bacteriostatic and bactericidal from the purple gland of the sea hare Aplysia dactylomela28 and the substrates determine the bactericidal or bacteriostatic effects. Another protein called achacin possesses a bacteriostatic effect that seems to be mediated through its oxidation of the L– amino acids lysine and/or arginine and the consequent subsequent production of hydrogen peroxide29. Another antimicrobial protein has been named as escapin from the Aplysia sp., which has been cloned and characterized. Thus this study reports the new protein Lolduvin-S with a potent LAO activity from melanin free ink of Loligo duvauceli. Lolduvin-S from the melanin free ink sample has been found to be more effective against the Gram positive bacteria, gram negative bacteria and the pathogenic yeast.

In an earlier study, a protein in low concentration with potent L-amino acid oxidase activity showing a broad antibacterial activity against Gram-positive and Gram-negative bacteria by H2O2 generation has been reported from the skin mucous of the rock fish30. In correlation with this, the MIC value is determined at low concentration range of 6.25µg/ml. A similar protein has also been isolated from two purple pigmented bacteria from Mediterranean sea whose enzymatic activity is potent in generating the hydrogen peroxide in the presence of Lamino acids31 . A BLAST search suggests that the cDNA encoded a novel antibacterial protein sharing identity with a number of L-amino acid oxidases is effective against MRSA strain32. In correlation with these reports, this study states that the new protein Lolduvin-S from Loligo duvauceli‘s ink possess a promising antibacterial property against the drug resistant pathogens. The bioactive protein Lolduvin-S also scores to possess a high antifungal activity against C.albicans with a MIC value of 12.5 µg/ml. The same property has been reported from novel proteins from snake venoms33. Four glycoproteins with similar activity have been studied from a shell less gastropod sea hare34. A similar protein called Aplysianin-A with antifungal activity has been isolated, cloned and characterized from the purple fluid released from a sea hare with LAO activity35. The antifungal activity of nine amphibian skin secretions has been reported to possess a similar protein with LAO activity36 . Several cell surface proteins with LAO activity have been isolated from marine phytoplanktons with potent antifungal activity37. In relation with these reports, Lolduvin-S from Loligo duvauceli‘s ink possesses a potent antifungal activity. In conclusion, Lolduvin–S, a new and novel protein from the ink of the Indian squid Loligo duvauceli with a potent antimicrobial and LAO activity, can be considered in near future as a valuable pharmaceutical antimicrobial agent from the marine habitat. However further characterization and structural analysis of the same is under progress to acquire a detailed understanding of this novel protein. Lolduvin-S being found to be bactericidal against the ESBL and MRSA pathogens, it can definitely aid in the control and emergence of drug resistant strains globally.

ACKNOWLEDGEMENT

We are thankful to Dr.Mehar Sultana.,M.Sc.,M.Phil.,Ph.D., Professor, Department of Zoology, Presidency College, Chennai, Tamilnadu for speciating the squid selected for our study.

 

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9. Gian Luigi, Elio Nisco, Gabriella, Paola and Anna Palumbo. Toxicity of melanin free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase. Biochem and Biophy Resear Comm 2003; 308: 293-299.

10. Takaya Y, Uchisawa H, Matsue H, Okusaki B. An investigation of the antitumour peptide form squid ink. Biol Pharm Bull 1994; 17: 846-849.

11. Charles Derby, Hsiuchin Yang, Paul Micah, Ko-Chun Ko and Phang. Cloning, characterization and expression of escapin, a broadly antimicrobial FAD – containing Lamino acid Oxidase from ink of the sea hare Aplysia californica. J of Expe Biol 2005; 208: 3609-3622.

12. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Estimation of proteins. J of Biol Chem1951; 193: 265-266.

13. Nagashima Y, Kikuchi N, Shimakura K, Shiomi K. Purification and characterization of an antibacterial protein in the skin secretion of rockfish. Comp Biochem Physiol 2003; 136: 63-71.

14. Anbalagan K. An introduction to electrophoresis. Laboratory Manual, Yercaud Biotech 1999; 1st Edition.

15. Pierce Laboratories. Technical Resource. Purify proteins from Polyacrylamide gels. Rockford, USA. www.piercenet.com.

16. MacHeroux P, Seth O, Bollschweiler C, Ghisla A. L-amino oxidase from the malayan pit Viper calloselasma rhodostoma comparative sequence analysis and characterization of active and inactive forms of the enzyme. Eur J Biochem 2001; 268: 1679-1686.

17. Nathan P, Law EJ, Murphy DF. A laboratory method for the selection of topical antimicrobial agents. Burns 1978; 4: 177 178.

18. Rios JL, Recio MC, Villar A. Screening methods for natural products with antimicrobial activity: a review of the literature. J Ethnopharma leaflets 1988; 23: 127-149.

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20. Nkere CK, Iroegbu C. Antibacterial screening of the root, seed and stembark extracts of Picralima nitida. Afri J of Biotech 2005; 4(6): 522-526.

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A Study by Humaira Tahir et al. entitled "Comparison of First Analgesic Demand after Major Surgeries of Obstetrics and Gynecology between Pre-Emptive Versus Intra-Operative Groups by Using Intravenous Paracetamol: A Cross-Sectional Study" is awarded Best Article for Vol 14 issue 14
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A Study by Mokhtar M Sh et al. entitled "Prevalence of Hospital Mortality of Critically Ill Elderly Patients" is awarded Best Article for Vol 14 issue 12
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A Study by Pandya S. et al. entitled "Acute and 28-Day Repeated Dose Subacute Toxicological Evaluation of Coroprotect Tablet in Rodents" is awarded Best Article for Vol 14 issue 10
A Study by Muhammad Zaki et al. entitled "Effect of Hemoglobin Level on the Severity of Acute Bronchiolitis in Children: A Case-Control Study" is awarded Best Article for Vol 14 issue 09
A Study by Vinita S & Ayushi S entitled "Role of Colour Doppler and Transvaginal Sonography for diagnosis of endometrial pathology in women presenting with Abnormal Uterine Bleeding" is awarded Best Article for Vol 14 issue 08
A Study by Prabhu A et al. entitled "Awareness of Common Eye Conditions among the ASHA (Accredited Social Health Activist) Workers in the Rural Communities of Udupi District- A Pilot Study" is awarded Best Article for Vol 14 issue 07
A Study by Divya MP et al. entitled "Non-Echoplanar Diffusion-Weighted Imaging and 3D Fiesta Magnetic Resonance Imaging Sequences with High Resolution Computed Tomography Temporal Bone in Assessment and Predicting the Outcome of Chronic Suppurative Otitis Media with Cholesteatoma" is awarded Best Article for Vol 14 issue 06
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A Study by Mbungu Mulaila AP et al. entitled "Ovarian Pregnancy in Kindu City, D.R. Congo - A Case Report" is awarded Best Article for Vol 14 issue 03
A Study by Maryam MJ et al. entitled "Evaluation Serum Chemerin and Visfatin Levels with Rheumatoid Arthritis: Possible Diagnostic Biomarkers" is awarded Best Article for Vol 14 issue 02
A Study by Shanthan KR et al. entitled "Comparison of Ultrasound Guided Versus Nerve Stimulator Guided Technique of Supraclavicular Brachial Plexus Block in Patients Undergoing Upper Limb Surgeries" is awarded Best Article for Vol 14 issue 01
A Study by Amol Sanap et al. entitled "The Outcome of Coxofemoral Bypass Using Cemented Bipolar Hemiarthroplasty in the Treatment of Unstable Intertrochanteric Fracture of Femur in a Rural Setup" is awarded Best Article Award of Vol 13 issue 24
A Study by Manoj KP et al. entitled "A Randomized Comparative Clinical Trial to Know the Efficacy of Ultrasound-Guided Transversus Abdominis Plane Block Against Multimodal Analgesia for Postoperative Analgesia Following Caesarean Section" is awarded Best Article Award of Vol 13 issue 23
A Study by Karimova II et al. entitled "Changes in the Activity of Intestinal Carbohydrases in Alloxan-Induced Diabetic Rats and Their Correction with Prenalon" is awarded Best Article of Vol 13 issue 22
A Study by Ashish B Roge et al. entitled "Development, Validation of RP-HPLC Method and GC MS Analysis of Desloratadine HCL and It’s Degradation Products" is awarded Best Article of Vol 13 issue 21
A Study by Isha Gaurav et al. entitled "Association of ABO Blood Group with Oral Cancer and Precancer – A Case-control Study" is awarded Best Article for Vol 13 issue 20
A Study by Amr Y. Zakaria et al. entitled "Single Nucleotide Polymorphisms of ATP-Binding Cassette Gene(ABCC3 rs4793665) affect High Dose Methotrexate-Induced Nephrotoxicity in Children with Osteosarcoma" is awarded Best Article for Vol 13 issue 19
A Study by Kholis Ernawati et al. entitled "The Utilization of Mobile-Based Information Technology in the Management of Dengue Fever in the Community Year 2019-2020: Systematic Review" is awarded Best Article for Vol 13 issue 18
A Study by Bhat Asifa et al. entitled "Efficacy of Modified Carbapenem Inactivation Method for Carbapenemase Detection and Comparative Evaluation with Polymerase Chain Reaction for the Identification of Carbapenemase Producing Klebsiella pneumonia Isolates" is awarded Best Article for Vol 13 issue 17
A Study by Gupta R. et al. entitled "A Clinical Study of Paediatric Tracheostomy: Our Experience in a Tertiary Care Hospital in North India" is awarded Best Article for Vol 13 issue 16
A Study by Chandran Anand et al. entitled "A Prospective Study on Assessment of Quality of Life of Patients Receiving Sorafenib for Hepatocellular Carcinoma" is awarded Best article for Vol 13 issue 15
A Study by Rosa PS et al. entitled "Emotional State Due to the Covid – 19 Pandemic in People Residing in a Vulnerable Area in North Lima" is awarded Best Article for Vol 13 issue 14
A Study by Suvarna Sunder J et al. entitled "Endodontic Revascularization of Necrotic Permanent Anterior Tooth with Platelet Rich Fibrin, Platelet Rich Plasma, and Blood Clot - A Comparative Study" is awarded Best Article for Vol 13 issue 13
A Study by Mona Isam Eldin Osman et al. entitled "Psychological Impact and Risk Factors of Sexual Abuse on Sudanese Children in Khartoum State" is awarded Best Article for Vol 13 issue 12
A Study by Khaw Ming Sheng & Sathiapriya Ramiah entitled "Web Based Suicide Prevention Application for Patients Suffering from Depression" is awarded Best Article for Vol 13 issue 11
A Study by Purushottam S. G. et al. entitled "Development of Fenofibrate Solid Dispersions for the Plausible Aqueous Solubility Augmentation of this BCS Class-II Drug" is awarded Best article for Vol 13 issue 10
A Study by Kumar S. et al. entitled "A Study on Clinical Spectrum, Laboratory Profile, Complications and Outcome of Pediatric Scrub Typhus Patients Admitted to an Intensive Care Unit from a Tertiary Care Hospital from Eastern India" is awarded Best Article for Vol 13 issue 09
A Study by Mardhiah Kamaruddin et al. entitled "The Pattern of Creatinine Clearance in Gestational and Chronic Hypertension Women from the Third Trimester to 12 Weeks Postpartum" is awarded Best Article for Vol 13 issue 08
A Study by Sarmila G. B. et al. entitled "Study to Compare the Efficacy of Orally Administered Melatonin and Clonidine for Attenuation of Hemodynamic Response During Laryngoscopy and Endotracheal Intubation in Gastrointestinal Surgeries" is awarded Best Article for Vol 13 issue 07
A Study by M. Muthu Uma Maheswari et al. entitled "A Study on C-reactive Protein and Liver Function Tests in Laboratory RT-PCR Positive Covid-19 Patients in a Tertiary Care Centre – A Retrospective Study" is awarded Best Article of Vol 13 issue 06 Special issue Modern approaches for diagnosis of COVID-19 and current status of awareness
A Study by Gainneos PD et al. entitled "A Comparative Evaluation of the Levels of Salivary IgA in HIV Affected Children and the Children of the General Population within the Age Group of 9 – 12 Years – A Cross-Sectional Study" is awarded Best Article of Vol 13 issue 05 Special issue on Recent Advances in Dentistry for better Oral Health
A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" is awarded Best Article of Vol 13 issue 06
A Study by Chen YY and Ghazali SRB entitled "Lifetime Trauma, posttraumatic stress disorder Symptoms and Early Adolescence Risk Factors for Poor Physical Health Outcome Among Malaysian Adolescents" is awarded Best Article of Vol 13 issue 04 Special issue on Current Updates in Plant Biology to Medicine to Healthcare Awareness in Malaysia
A Study by Kumari PM et al. entitled "Study to Evaluate the Adverse Drug Reactions in a Tertiary Care Teaching Hospital in Tamilnadu - A Cross-Sectional Study" is awarded Best Article for Vol 13 issue 05
A Study by Anu et al. entitled "Effectiveness of Cytological Scoring Systems for Evaluation of Breast Lesion Cytology with its Histopathological Correlation" is awarded Best Article of Vol 13 issue 04
A Study by Sharipov R. Kh. et al. entitled "Interaction of Correction of Lipid Peroxidation Disorders with Oxibral" is awarded Best Article of Vol 13 issue 03
A Study by Tarek Elwakil et al. entitled "Led Light Photobiomodulation Effect on Wound Healing Combined with Phenytoin in Mice Model" is awarded Best Article of Vol 13 issue 02
A Study by Mohita Ray et al. entitled "Accuracy of Intra-Operative Frozen Section Consultation of Gastrointestinal Biopsy Samples in Correlation with the Final Histopathological Diagnosis" is awarded Best Article for Vol 13 issue 01
A Study by Badritdinova MN et al. entitled "Peculiarities of a Pain in Patients with Ischemic Heart Disease in the Presence of Individual Combines of the Metabolic Syndrome" is awarded Best Article for Vol 12 issue 24
A Study by Sindhu Priya E S et al. entitled "Neuroprotective activity of Pyrazolone Derivatives Against Paraquat-induced Oxidative Stress and Locomotor Impairment in Drosophila melanogaster" is awarded Best Article for Vol 12 issue 23
A Study by Habiba Suhail et al. entitled "Effect of Majoon Murmakki in Dysmenorrhoea (Usre Tams): A Standard Controlled Clinical Study" is awarded Best Article for Vol 12 issue 22
A Study by Ghaffar UB et al. entitled "Correlation between Height and Foot Length in Saudi Population in Majmaah, Saudi Arabia" is awarded Best Article for Vol 12 issue 21
A Study by Siti Sarah Binti Maidin entitled "Sleep Well: Mobile Application to Address Sleeping Problems" is awarded Best Article for Vol 12 issue 20
A Study by Avijit Singh"Comparison of Post Operative Clinical Outcomes Between “Made in India” TTK Chitra Mechanical Heart Valve Versus St Jude Mechanical Heart Valve in Valve Replacement Surgery" is awarded Best Article for Vol 12 issue 19
A Study by Sonali Banerjee and Mary Mathews N. entitled "Exploring Quality of Life and Perceived Experiences Among Couples Undergoing Fertility Treatment in Western India: A Mixed Methodology" is awarded Best Article for Vol 12 issue 18
A Study by Jabbar Desai et al. entitled "Prevalence of Obstructive Airway Disease in Patients with Ischemic Heart Disease and Hypertension" is awarded Best Article for Vol 12 issue 17
A Study by Juna Byun et al. entitled "Study on Difference in Coronavirus-19 Related Anxiety between Face-to-face and Non-face-to-face Classes among University Students in South Korea" is awarded Best Article for Vol 12 issue 16
A Study by Sudha Ramachandra & Vinay Chavan entitled "Enhanced-Hybrid-Age Layered Population Structure (E-Hybrid-ALPS): A Genetic Algorithm with Adaptive Crossover for Molecular Docking Studies of Drug Discovery Process" is awarded Best article for Vol 12 issue 15
A Study by Varsha M. Shindhe et al. entitled "A Study on Effect of Smokeless Tobacco on Pulmonary Function Tests in Class IV Workers of USM-KLE (Universiti Sains Malaysia-Karnataka Lingayat Education Society) International Medical Programme, Belagavi" is awarded Best article of Vol 12 issue 14, July 2020
A study by Amruta Choudhary et al. entitled "Family Planning Knowledge, Attitude and Practice Among Women of Reproductive Age from Rural Area of Central India" is awarded Best Article for special issue "Modern Therapeutics Applications"
A study by Raunak Das entitled "Study of Cardiovascular Dysfunctions in Interstitial Lung Diseas epatients by Correlating the Levels of Serum NT PRO BNP and Microalbuminuria (Biomarkers of Cardiovascular Dysfunction) with Echocardiographic, Bronchoscopic and HighResolution Computed Tomography Findings of These ILD Patients" is awarded Best Article of Vol 12 issue 13 
A Study by Kannamani Ramasamy et al. entitled "COVID-19 Situation at Chennai City – Forecasting for the Better Pandemic Management" is awarded best article for  Vol 12 issue 12
A Study by Muhammet Lutfi SELCUK and Fatma entitled "Distinction of Gray and White Matter for Some Histological Staining Methods in New Zealand Rabbit's Brain" is awarded best article for  Vol 12 issue 11
A Study by Anamul Haq et al. entitled "Etiology of Abnormal Uterine Bleeding in Adolescents – Emphasis Upon Polycystic Ovarian Syndrome" is awarded best article for  Vol 12 issue 10
A Study by entitled "Estimation of Reference Interval of Serum Progesterone During Three Trimesters of Normal Pregnancy in a Tertiary Care Hospital of Kolkata" is awarded best article for  Vol 12 issue 09
A Study by Ilona Gracie De Souza & Pavan Kumar G. entitled "Effect of Releasing Myofascial Chain in Patients with Patellofemoral Pain Syndrome - A Randomized Clinical Trial" is awarded best article for  Vol 12 issue 08
A Study by Virendra Atam et. al. entitled "Clinical Profile and Short - Term Mortality Predictors in Acute Stroke with Emphasis on Stress Hyperglycemia and THRIVE Score : An Observational Study" is awarded best article for  Vol 12 issue 07
A Study by K. Krupashree et. al. entitled "Protective Effects of Picrorhizakurroa Against Fumonisin B1 Induced Hepatotoxicity in Mice" is awarded best article for issue Vol 10 issue 20
A study by Mithun K.P. et al "Larvicidal Activity of Crude Solanum Nigrum Leaf and Berries Extract Against Dengue Vector-Aedesaegypti" is awarded Best Article for Vol 10 issue 14 of IJCRR
A study by Asha Menon "Women in Child Care and Early Education: Truly Nontraditional Work" is awarded Best Article for Vol 10 issue 13
A study by Deep J. M. "Prevalence of Molar-Incisor Hypomineralization in 7-13 Years Old Children of Biratnagar, Nepal: A Cross Sectional Study" is awarded Best Article for Vol 10 issue 11 of IJCRR
A review by Chitra et al to analyse relation between Obesity and Type 2 diabetes is awarded 'Best Article' for Vol 10 issue 10 by IJCRR. 
A study by Karanpreet et al "Pregnancy Induced Hypertension: A Study on Its Multisystem Involvement" is given Best Paper Award for Vol 10 issue 09

List of Awardees

A Study by Ese Anibor et al. "Evaluation of Temporomandibular Joint Disorders Among Delta State University Students in Abraka, Nigeria" from Vol 13 issue 16 received Emerging Researcher Award


A Study by Alkhansa Mahmoud et al. entitled "mRNA Expression of Somatostatin Receptors (1-5) in MCF7 and MDA-MB231 Breast Cancer Cells" from Vol 13 issue 06 received Emerging Researcher Award


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International Journal of Current Research and Review (IJCRR) provides platform for researchers to publish and discuss their original research and review work. IJCRR can not be held responsible for views, opinions and written statements of researchers published in this journal

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