Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241914EnglishN2017July28General SciencesNovel Application of Reversed Gel Electrophoresis: Reuse of DNA Molecular Weight Marker
English0102Suhasa G.English Savithri BhatEnglishGel electrophoresis is used in nucleic acid analysis and makes use of DNA/RNA molecular weight marker for size comparison. These size markers are used only once per electrophoresis and finally discarded along with the gel. In the present study, a reversed current is applied in agarose gel electrophoresis to reuse the DNA molecular weight marker, thus cutting down the cost involved in molecular experiments.
EnglishAgarose gel electrophoresis, DNA ladder, Nucleic acidAgarose gel electrophoresis is a method for separating nucleic acids based on size by comparing DNA or RNA against standard molecular weight marker1. These markers are also called as nucleic acid ladders which contain various DNA or RNA of specific length. Agarose gel electrophoresis, which is mainly used for restriction fragment analysis and PCR product analysis, uses DNA ladder for size comparison. The agarose gel is loaded with DNA test sample and DNA molecular weight marker and when electric current is passed DNA moves towards anode. After the completion of electrophoresis the aligned position of DNA test sample is compared with the resolved DNA ladder and the size of DNA in test sample is determined. The resolved DNA ladder is not reused for another electrophoresis experiment. Reversed electrophoresis had been used to purify protein from polyacrylamide gel2. Rapid methods for isolation of peptides from sodium dodecyl sulphate (SDS) containing polyacrylamide gels have been applied3 and been used for purification of chymotrypsin inhibitor isolforms4.
In agarose gel electrophoresis, after the separation of DNA, the sample well corresponding to resolved DNA ladder is sealed with agarose. By changing the polarity of electrodes or placing the agarose gel with the wells facing towards anode results in movement of resolved DNA ladder converging back as a single band in the sealed well of agarose gel. After 15-20 minutes of reversed electrophoresis, the gel is kept for 20 minutes in a vessel containing 0.5 µg/ml ethidium bromide (so that the DNA that has lost the stain is re-stained and is clearly visible under UV light). The stained gel is kept back in electrophoresis tank and reversed electrophoresis is carried out until the DNA ladder appears back in its sealed well. The gel slice containing DNA ladder present as a single band is removed and the gel slice is stored in buffer in refrigerator until its next use. The gel extraction using spin column can be used to purify DNA ladder from gel slice but is costlier. Therefore, the stored gel slice, when required, is placed in fresh well of agarose gel and the well is sealed with agarose. The DNA test samples that are to be analyzed are loaded in the wells of other lanes. The gel electrophoresis again resolves the DNA ladder producing bands with almost the same intensity as the fresh DNA ladder (Fig.1). The average cost of commercially available 100bp DNA ladder is Rs.40/- per gel lane. The reuse of DNA ladder helps in cutting down its cost by 50% per gel lane and costs Rs.20/- per gel lane.
It was observed that the gel slice containing the DNA ladder can be stored for a minimum of fifteen days without much loss in band intensity as seen after its re-electrophoresis.
Conclusion:
The reversed gel electrophoresis discussed in this paper utilizes the Tris-acetate-EDTA buffer previously used to resolve DNA ladder. The electric current polarity was reversed by interchanging the chords connecting the platinum electrodes. The gel plug containing the converged DNA ladder was removed using scalpel. Hence there is no requirement of additional reagents or time consuming steps. We conclude that the reuse of 100 bp DNA ladder is a cost-effective method that can be utilised in research laboratories.
Gel electrophoresis was run at 100V (5.56V/cm) for 50 minutes using 3% agarose gel and 0.5X TAE. RDL, Reused DNA ladder, 100bp DNA ladder resolved again from agarose plug which was collected previously by reversed gel electrophoresis, FDL, Fresh DNA ladder, Fresh 100bp DNA ladder.
Acknowledgements:
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Source of funding:
The present work was carried out with the help of financial support by TEQIP, BMSCE (Grant No.16-5/2013-TS.VII (Pt.)) and VGST, KFIST-1 (GRD 361).
Conflict of Interest:
The authors don’t have any conflict of interest
Englishhttp://ijcrr.com/abstract.php?article_id=1771http://ijcrr.com/article_html.php?did=17711. Green MR, Sambrook J. Analysis of DNA. In: Molecular Cloning a laboratory manual, 4th ed. CSH press, USA 2012.
2. Otto M, Snejdarkova M. A simple and rapid method for the quantitative isolation of proteins from polyacrylamide gels. Analytical Biochemistry 1981; 111: 111-14.
3. Hartvig, IM. A simple and rapid method for the isolation of peptides from sodium dodecyl sulfate-containing polyacrylamide gels. Analytical Biochemistry 1982; 121: 215-17.
4. Lim JI, Bae BN, Jhun BS, Kang IS, Kim SJ. A Simple Preparative Polyacrylamide Gel Electrophoresis for the Purification of Chymotrypsin Inhibitor Isoforms from Ganoderma lucidum. Bulletin of the Korean Chemical Society 2003; 24: 1531-34.
Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241914EnglishN2017July28General SciencesGreen Synthesis and Characterization of Polyvinyl Alcohol Embedded Silver Nanoparticles Using Simul (Bombax ceiba) Flower Extract
English0306Pinaki MandalEnglishAim: Plants biomass extract have very important role for the synthesis of different nanoparticles due to its reducing capability, availability and nontoxicity. The present study reports a very easy and green synthesis of poly (vinyl alcohol) stabilized silver nanoparticles using Simul (Bombax ceiba) flower extract.
Methodology: The aqueous extract of Simul (Bombax ceiba) flower was used for this bio-reduction. When PVA supported aqueous silver ions exposed to aqueous Simul flower extract, silver ions were reduced and result was green synthesis of silver nanoparticles.
Results: The produced silver nanoparticles were characterised by different technique which includes UV–vis spectroscopy and transmission electron microscopy (TEM). The highest peak at 470 nm in UV-visible spectrum due to surface Plasmon resonance confirmed the presence of silver nanoparticles. TEM analysis showed the spherical and irregular particles shape and size ranging from 20 to 100 nm.
Conclusion: This single-pot method for synthesis of silver nanoparticle is eco-friendly and cost effective than other hazardous chemical methods. Different therapeutic use of PVA-supported silver nanoparticles synthesized from Simul flower, otherwise thrown away as ineffective material into environment is note worthy.
EnglishSimul, Silver nanoparticle, Polyvinyl alcohol, Green synthesisINTRODUCTION
Now a days, research on metallic nanoparticles (NPs) arean important field in nanotechnology which deals with synthesis and manipulation of particle’s size ranging from approximately 1 to 100 nm in size (Ahmed S. et al., 2016; Yehia et al., 2014). Because of their particular size and morphology nanoparticles exhibits novel chemical, biological and physical properties than their parent materials and are found to be interesting candidates for various applications which includes antimicrobial activity, biosencing particularly in biomedical science (Maiti S. et al., 2014: Padalia H. et al., 2015; Ahmad T. et al., 2013; Okafor F. et al., 2013; Abou et al., 2010) However, particle aggregation remarkably deteriorates their properties. Recently different organic polymer like polyvinyl alcohol (PVA), polyacrylic acid, polyarylesters, chitosan, and poly-acrylonitrile can be used to support, stabilize as well as surface modification, distribution of the nanoparticles (Gaddy G. A. et al., 2004; Rifai S. et al., 2006; Valente, J. F. A., et al., 2013; Chandran S., et al., 2016).The functional groups present in polymeric material plays vital role to efficiently immobilize nanoparticles by different factors like Vander Waals force, electrostatic force, hydrogen or covalent bonds (Sagitha P. et al., 2016).Polyvinyl alcohol has easy acceptability being biodegradable, water soluble, inexpensive and low-toxicity to prepare polyvinyl alcoholnanoparticles (PVA-NPs) hybrid. Synthesis of PVA-AgNPs is of much interest to the scientific community because of their wide range of applications. There are many physical and chemical methods for the synthesis of PVA-AgNPs. However most of the methods are quite expensive and potentially hazardous to the environment which involves use of toxic and perilous chemicals that are responsible for various biological risks (Ahmed S. et al., 2016; Prozorova G. F et al., 2014). So, there is a need to develop high-yield, low cost, nontoxic and environmentally friendly procedures. In such a situation, biological approach appears to be very appropriate. Natural materials like plant (flower, leaf, bark, fruit etc.) extract or plant biomass, microorganisms for the production of silver nanoparticles could be an alternative to chemical and physical methods in an eco-friendly manner(Reddy G. A. K. et al., 2012) Simul tree (Bombax ceiba) is very useful tree and is found throughout the Asia. Simul commonly known as ‘cotton tree’ because it produces a white fibre like cotton capsule. It is generally tall straight tree, its leaves are deciduous in winter, red coloured flower with 5 petals appear in spring season. Different parts of this tree have meditional properties. In Ayurveda it is referred as aphrodisiac, astringent, antidiarrheal, antidysenteric, antimicrobial diuretic, alterative, antipyretic and tonic. It is used in treatment of asthma, diarrhoea, wound, leucorrhoea, anaemia, seminal disorders and skin problems (Asolkar L. V. et al., 1992). A large number of phytochemicals have been found from Simul flower that include glycosides carbohydrate, protein, flavonoids, etc. Here the advantages for synthesis of PVA-AgNPs using Bombax ceiba flower is that their easy availability, non-toxicity and presence of a broad variability of biomolecules which may aid in reduction process (Joshi K. R. et. al., 2013) This article reports the simple green method for synthesis of polyvinyl alcohol supported silver nanoparticles (PVAAgNPs) using flower extract of Bombax ceiba. According to my knowledge this is the first report for the synthesis of PVA-AgNPs hybrid using Simul flower extract.
MATERIALS AND METHODS
Preparation of flower extract
Fresh flowers of Bombax ceiba growing in road side of Bankura district, West Bengal, India, were collected (Figure 1). Fresh flowers were washed extensively with water followed by final wash with double distilled water. Washed flower were dried in sunlight and after grinding 5 g of flower powder was mixed with 100 ml double distilled water in 250 ml Borosil beaker and heated for 60 minutes. Then the extract was filtered through Whatman filter paper (pore size >0.5μm), collected and stored in refrigerator(40 C) touse within 1 week.
Preparation of metal solution
Initially 1M AgNO3 solution was prepared (4.25 g silver nitrate was dissolved in 25 ml double distilled water).From it 0.25 M AgNO3 solution was prepared.
Synthesis of nanoparticles (AgNPs)
The aqueous Simul flower extract had been used for the bioreduction process. At first 0.5 ml of the flower extract was mixed with 10% 0.5 ml PVA solution and then 3 ml of 0.25M AgNO3 solution was mixed and kept at room temperature for 1 hour. Colour of the solution was changed from very light yellow to dark brownish indicating the formation of silver nanoparticles. Characterization of SNPs by UV-vis spectroscopy and TEM Synthesis of AgNPs was monitored using a Shimadzu (UV1800) double-beam spectrophotometer. The absorption spectrum in the range of 400 to700 nm of the reaction medium after diluting a small aliquot of sample and crude water extract. TEM (Transmission electron microscopy) analysis was done to visualize the shapes well as to measure the diameter of the bio-synthesized silver nanoparticles. Nanoparticles were screened in high resolution JEOL JEM1400 plus microscope operating at an accelerated voltage of 120 kV.
RESULTS
Characterization of nanoparticles by Colour change, UV–Visible spectra and TEM
Upon addition of flower extract to the solution of PVA-AgNO3, the colour appeared as dark brown within 1 h of incubation at room temperature (Figure 2) indicating the formation of nanosilver. The formation of AgNPs occurs from few minutes to hour and corresponding UV–Visible absorption spectra of AgNPs were recorded which shown in Figure3. The shape and size distribution of the synthesized AgNPs were characterized by TEM study (Figure 4).
DISCUSSION
The bio-reduction of PVA supported silver ions to PVA-AgNPs using Simul flower extract was understood visually by
colour change of the reaction mixture from very light yellow to dark brown (Figure 2). The intensity of colour increases with increase in time which indicates the reduction of more silver ions and more silver nanoparticles formed. The characteristic dark brown colour of silver provided a convenient spectroscopic signature to indicate nanoparticles formation. The formation of silver nanoparticles can be detected by UV-Visible spectroscopy by analyzing its characteristic surface plasmon resonance band (SPR) due to the combined vibration of electrons of metal nanoparticles in resonance with the light wave (Rajkumar G. et al., 2012). The maximum absorbance peak at 470 nm in the UV-absorption spectrum, confirmed the presence of surface plasmon resonance peak of silver nanoparticles (Figure 3). The intensity of the absorption peak increases steadily with time indicates that the intensity of colour was directly proportional to the amount of AgNPs produced and the results corresponds to the earlier studies which reports green synthesis of silver nanoparticles showing absorption band 420-480nm (Rajana R. et al., 2015) The polymer supported nanoparticles are imaged using transmission electron microscopy (Figure 4) to further confirm the generation of SPR band in the UV-visible spectrum for AgNPs. The photograph of TEM shows formation of non-agglomerated silver nanoparticles with spherical and irregular particles shape and size ranging from 20 to 100 nm.
CONCLUSION
Using Simul flower extract a simple, low-cost, eco-friendly one-pot synthesis was carried out to prepare Polyvinyl supported silver nanoparticles at room temperature. Biosynthesized PVA-AgNPswas characterized from the characteristic surface plasmon resonance peak obtained from the UV-Visible spectroscopic studies. Morphology studies were done using transmission electron microscope. This synthesis method and reaction conditions lead to the formation of silver nanoparticles of 20-100 nm in size with spherical and irregular particles shape. The synthesized PVA-silver nanoparticle hybrid can show new pathways in various fields like bio-sensing, antibacterial, water purification, antifungal, anticancer activity and specially drug delivery systems.
ACKNOWLEDGEMENTS
Author acknowledges the immense help received from the scholars whose articles are cited and included as references in this manuscript. The author is also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. The author is also grateful to Dr. Samaresh Ghosh, Dept. of chemistry, Bankura Sammilani College, Bankura, for his support in this work.
Conflict of Interest The author has declared that no conflict of interest exists.
Source of Funding Nil
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241914EnglishN2017July28General SciencesGrowth Promoting and Probiotic Potential of the Endophytic Bacterium Rhodococcus globerulus colonizing the Medicinal Plant Plectranthus amboinicus (Lour.) Spreng
English0713R.M. MurugappanEnglish S. Benazir begumEnglish C. UshaEnglish S. Lok kirubaharEnglish M. KarthikeyanEnglishObjectives: It was intended to isolate endophytic bacteria from Plectranthus amboinicus leaves to elucidate the plant growth promotion potential.
Methodology: Phytochemical constituents of Plectranthus amboinicus leaves were analyzed following standard procedures. Endophytic bacteria were isolated in nitrogen limited medium after surface sterilization process as described by Coombs & Franco (2003) and identified by biochemical, molecular analyses. Plant growth promotion and quorum quenching potential of the isolate was determined as described by Patel et al. (2011). Probiotic potential of the isolates was elucidated following a series of assays (Ahire et al. 2011).
Results: Phytochemical investigation of the leaves revealed the presence of alkaloids, flavonoids and terpenoids. An endophytic bacterium with probiotic properties was isolated and identified as Rhodococcus globerulus. Plant growth promotion (PGP) traits such as phosphate solubilization, IAA production and ACC deaminase activity of R. globerulus was estimated to be 37.3 μg ml-1, 36.7 μg ml-1 and 35.00 ± 1.2 nmol α-ketobutyrate mg-1 h-1 respectively at 60 h of incubation. The isolate was found to produce trihydroxamate siderophores with hexadentate ligands. Presence of the bacterium within the leaf tissues was confirmed by SEM imaging. R. globerulus was found to produce quorum sensing inhibitor (QSI) molecules. Endophytic isolate was also found to possess probiotic potential.
Conclusion: The results illustrate that the plant-associated microbiome serve as a repertoire of metabolic products that facilitate nutritional and defense pathway of the host plant. Probiotic properties of the endophyte confer an added advantage to the therapeutic potential of the plant P. amboinicus.
EnglishPlant growth promotion, Plectranthus amboinicus, probiotic, quorum sensing, Rhodococcus globerulus, siderophoresIntroduction
Plant tissues constitute a nutrient rich niche for metabolically versatile microorganisms called endophytes. Bacon and White [1] defined endophytes as “microbes that colonize, living, internal tissues of plants without causing any immediate, overt negative effects”. Unlike a random collection, endophytic microbes are specific and inextricably associated with the host plant [2]. Endophytes stimulate plant growth by nitrogen fixation, phosphate solubilization, iron chelation, phytohormone and 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme synthesis and / or by the prevention of pathogen infections [3]. Endophytes are reported to attenuate microbial pathogenicity by the synthesis of quorum sensing (QS) inhibitors [4].
Due to the long co-existence, there is a possibility of horizontal gene transfer which facilitates the endophytes to synthesize bioactive compounds of plant origin [5]. For example, piperine was isolated from endophytic fungus Periconia sp. colonizing the host plant Piper longum L. [6].
Despite their innate importance, endophytic microbial community in medicinal plants remains largely uncharacterized. Plectranthus amboinicus (Lour.) Spreng (Lamiaceae) a large succulent aromatic perennial herb commonly known as Indian borage is a native species of Asia. Leaves of P. amboinicus are taken orally for the treatment of respiratory infections, digestive diseases, mumps and wounds [7]. Screening the probiotic potential of the microbes associated ensures the safety in the uptake of P. amboinicus leaves for disease prevention.
Synthetic antioxidants and antimicrobials in use are reported to possess various side effects. Therefore, the search for natural antioxidants, especially of plant origin has attracted great attention. Medicinal plants represent a potential source of antioxidants that are more effective and less toxic.
Although there is a plethora of information on the medicinal properties of P. amboinicus, knowledge on the diversity, plant growth promotion and probiotic potential of its endophytic bacteria are limited. Therefore, the present study was intended to isolate endophytic bacteria from the leaves of P. amboinicus and to assess their growth promotion activities.
Materials and methods
Screening of phytochemical constituents
Fresh and healthy leaves of Plectranthus amboinicus were collected, washed and air-dried. Dried leaves (10 gm) were powdered and extracted with 10ml of milli-Q water by boiling at 45 ?C for 30 min. The extract was filtered and concentrated in a rotary evaporator (IKA RV-10, Germany) at 45 ?C under reduced pressure. The concentrate was subjected to qualitative phytochemical analyses following standard procedures [8].
Antioxidant and antimicrobial activity
Antioxidant potential of the leaf extract was measured by DPPH (2, 2-Diphenyl-1-picrylhydrazyl) method [9]. Susceptibility of bacterial pathogens to different concentration (20, 40, 60, 80 and 100 μg/ml) of leaf extract was determined by well diffusion method. Dose-response influence of aqueous leaf extract on pathogenic strains was calculated using prism software (ver. 6.04).
Isolation and identification of endophytic bacteria
For the isolation of endophytic bacteria, P. amboinicus leaves were cut into small discs (0.5 cm dia.) using sterile cork borer. Leaf discs were surface sterilized following the five step procedure described by Tiwari et al. [10]. Surface-sterilized leaves were air dried, triturated with phosphate buffered saline, serially diluted and spread onto the nutrient agar medium free of inorganic nitrogen. The plates were incubated for 48 h at 30 ºC. The endophytic bacteria were identified by conventional biochemical characteristics following Bergey’s Manual of Systemic Bacteriology [11] as well as by 16S rRNA gene sequencing by standard procedures [12]. Phylogenetic analysis was performed using Molecular Evolutionary Genetics Analysis software package (MEGA ver. 4.0) after multiple alignments of data by Clustal X.
Plant growth promotion (PGP) traits
Phosphate solubilization and indole acetic acid production
Phosphate solubilizing ability of the isolate was determined qualitatively and quantitatively by stannous chloride method [13] using Pikovskaya medium with 0.5 % tricalcium phosphate [Ca3 (PO4)2]. Indole acetic acid (IAA) production by the isolate on the utilization of L- tryptophan was determined using Salkowski reagent [14]. IAA production by the isolate was also confirmed by HPLC analysis.
ACC deaminase enzyme activity
ACC deaminase activity of the isolate was determined following the method of El-Tarabily [15]. Using Dworkin–Foster (DF) salts minimal medium amended with 3 mM of 1-aminocyclopropane-1-carboxylic acid (ACC) as a sole nitrogen source. ACC production was quantified by measuring the absorbance at 540 nm against a standard curve of α- ketobutyrate (Sigma, USA).
Screening of siderophore production
Siderophore production by the isolate was determined by FeCl3 test and chrome azurol sulphonate (CAS) agar plate method [16]. The amount of siderophore in the culture supernatant was quantified at different intervals by Chrome azurol sulphonate shuttle assay. Furthermore, the culture supernatant was subjected to Csaky, Arnow and Vogel’s assay to ascertain the nature of siderophore produced. Siderophore was further distinguished into mono-, di- or trihydroxamates based on the absorption maxima and electrophoretic mobility [17]. The iron-binding property of siderophore was determined by the color reaction at different pH values.
Production of HCN and ammonia
The isolate was screened for hydrogen cyanide production following the standard procedures [18]. Ammonia producing ability of the isolate was determined by the development of yellow color on the addition of Nessler’s reagent.
Screening of anti-quorum sensing activity
Anti-quorum sensing activity of endophytic bacteria against the reporter strain Chromobacterium violaceum (MTCC 2656) was determined following the procedure described by Mani et al. [19]. Mid log phase culture (18 h) of C. violaceum was inoculated in LB medium containing different concentration (25, 50, 75 and 100 μl) of endophytic bacterial culture filtrate. A control without culture filtrate was maintained. Culture tubes were incubated with shaking at 30ºC for 48 h. After incubation, 2 ml of culture was centrifuged at 8000 rpm for 10 min to precipitate the insoluble violacein and 2 ml of DMSO was added to the precipitate and vortexed (30 sec) to solubilize the violacein. The extracted violacein was quantified using a UV-vis spectrophotometer (OD585). The percentage of violacein inhibition was calculated by following the formula:
Percentage of violacein inhibition = (control OD585nm - test OD585nm / Control OD585nm) × 100.
Plant inoculation assay
For plant inoculation assay, shoot tips (5 inch) of healthy P. amboinicus with a pair of leaf nodes were excised using a scalpel, surface sterilized with 5 % sodium hypochlorite for 3 min and rinsed repeatedly with sterilized distilled water. Surface-sterilized shoot cuttings were immersed in endophytic bacterial cell suspension (107 CFU per ml of phosphate buffer pH 7.0) for 1 h, subsequently a control was maintained by immersing in sterile water. Treated and control shoot cuttings were planted in plastic pot (10×15 cm). Five replicates were maintained. The plants were grown in a greenhouse at 37ºC with 16 h light: 8 h darkness photoperiod. The plants were watered daily with sterile deionised water. After 3 weeks, the plants were uprooted and growth parameters such as shoot and root length and number of leaves were measured. The presence of endophytes within the leaf tissues was established by scanning electron microscopic (SEM) imaging.
Screening for probiotic properties
Acid and bile salt tolerance
A probiotic microorganism must overcome physical and chemical barriers in the gastrointestinal tract. Therefore, in this study acid tolerance property of the isolate was determined following the standard procedure [20]. The isolate was grown in nutrient broth for 24 h at 30ºC. Cells were harvested and re-suspended in 1 ml of sterile phosphate buffered saline (PBS) at different pH for various time intervals (0, 60, 120 and 180 min) and transferred to nutrient broth. The growth was measured at A620nm after 24 h. For bile salt tolerance, one ml (3×107 cells ml-1) of the isolate was inoculated on a nutrient broth supplemented with different concentration of oxgall (w/v) and the growth of the isolate was measured (A620nm) at different time intervals. Auto-aggregation was calculated by measuring the absorbance (660 nm) of the bacterial suspension in PBS (pH7.3) at different time intervals as described by Ahire et al. [21]. Antibiotic susceptibility and hemolytic ability of the isolate was determined following standard procedure.
Results
Phytochemical constituents
Qualitative determination of phytochemical constituents in the leaves of Plectranthus amboinicus revealed the presence of tannins, flavanoids, saponins, alkaloids, cardiac glycosides and terpenoids (Table 1). Absence of steroids and acidic compounds in the leaf extract are notable.
Antioxidant activity
The leaf extract of P. amboinicus showed a concentration dependent free radical scavenging activity with an EC50 value of 65.26 μg/ml. Maximum radical scavenging activity (61.06 %) was observed at 80 μg/ml. However, scavenging activity of ascorbic acid, a known antioxidant, which is used as a positive control in this study, was greater than that of leaf extract.
Antimicrobial activity
Aqueous leaf extract of P. amboinicus was found to inhibit the growth of the pathogenic microorganism viz., S.aureus, E.coli, M.luteus, P.aeruginosa, B.subtilis and S.typhi. Dose response curves illustrated that the pathogen inhibition was linearly associated with the concentration of the leaf extract (Fig.1) Inhibitory efficiency was found to be more effective against E. coli and M. luteus.
Isolation and identification of endophytic bacteria
Two phenotypically different endophytic bacterial strains were isolated from the leaves of P.amboinicus. The isolates were identified as Pseudomonas sp. and Rhodococcus sp. Of the two, Rhodococcus sp. was subjected to further analyses based on its non-haemolytic and catalase positive response. Based on 16 s rRNA gene sequencing (accession number KR013744) and phylogenetic analysis (Fig.2) the isolate was confirmed as Rhodococcus globerulus.
In vitro screening of Rhodococcus globerulus for PGP traits
Phosphate solubilization and indole acetic acid production by the isolate was determined qualitatively and quantitatively. Appearance of halo zones on Pikovskaya agar indicates phosphate solubilizing ability of the isolate (results not shown). The level of soluble phosphate in the medium was found to be 37.33μgml-1 at 60 h of incubation. In the present study, the isolate was found to solubilize the inorganic phosphate with the concomitant decline in medium pH (Fig. 3). Biosynthesis of 3-indole acetic acid by the isolate was estimated to be 6.33, 14.67, 21.33, 33.67, 36.69 and 28.67 μgml-1 at 12, 24, 36, 48, 60 and 72 h of incubation, respectively. HPLC chromatograms of the IAA produced by the isolate was compared with the standard (retention time 2.62) and presented in Fig. 4. Growth of the isolate in DF medium supplemented with ACC confirms ACC deaminase enzyme activity. Quantitative determination revealed high ACC deaminase activity (35.00 ±1.2 nmol α-ketobutyrate mg-1 h-1) by the isolate (Table 2).
The positive results with FeCl3 test, CAS assay and development of orange halos in CAS agar plate confirm siderophore production by the isolate (results not shown). Maximum siderophore production was observed only after 60 h of incubation and it was found to be 42.23 ± 0.67 % units. Absorbance maximum at 404 nm indicates hydroxamate nature of the siderophore produced and it was further confirmed by positive Csaky test. A narrow shift in λ-max (up to 8 nm) with different pH and electrophoretic mobility results indicate trihydroxamate nature of the siderophore (Table 3). The isolate exhibited other plant growth promotion traits such as HCN and ammonia production.
Anti-quorum sensing activity
In this study, quorum quenching ability of the R. globerulus was confirmed by the appearance of opaque zone on the periphery of reporter strain C. violaceum colonies (Fig. 5a). Violacein production was found to be decreased with increasing concentration of R. globerulus (Fig. 5b) culture filtrate.
Plant inoculation studies
Biopriming of P. amboinicus shoot with endophytic bacteria R. globerulus enhanced plant growth (Fig. 6). SEM imaging confirmed the colonization of isolate within the leaf tissues (Fig. 7).
Probiotic properties
R. globerulus was found to be viable at high acidic conditions (pH 2). Percent survival of R. globerulus after 3h of exposure to pH 2 and pH 4 was found to be 46.83 and 87.65 respectively (results not shown). In addition to acid tolerance, the isolate was able to survive at 2-4% concentration of bile salt. R. globerulus was found to be susceptible to tetracyclin, kanamycin, gentamycin and erythromycin but resistant to penicillin and rifampicin.
Discussion
Phytochemical screening ascertains the leaves of P. amboinicus are a rich source of tannins, flavanoids, saponins, alkaloids, cardiac glycosides and terpenoids (Table 1). Results on the qualitative analyses of phytochemicals are consistent with the available literature. Tannins and terpenoids are known to possess antimicrobial, anti-inflammatory and immunomodulatory properties [22]. Absence of steroid reveals the advantage of using P. amboinicus leaves in disease treatment. Antioxidant potential of the leaf extract might be due to the presence of phenolic compounds. The result obtained on free radical scavenging activity falls in line with the findings of Yadav et al. [23].
P. amboinicus leaf extract found to inhibit the growth of bacterial pathogens (Fig. 1). Neuwinger [24] reported the efficiency of Plectranthus sp. leaf extract in the prevention of microbial infections. Screening of surface sterilized leaf tissue of P. amboinicus revealed the presence of Pseudomonas sp. and Rhodococcus sp. Occurrence of Rhodococcus sp. within the leaf tissue of medicinal plants was reported in earlier studies [25]. It is still not clear whether plants benefit from an endophyte or if the advantage is higher for the bacteria to become endophyte. However it is evident from the results, the benefits conferred by endophytes are more conspicuous by growth promotion.
Growth promotion traits like indole acetic acid production, ACC deaminase, siderophore, HCN, ammonia production and phosphate solubilization confers the benefit of endophytic colonization of R. globerulus within the tissues of P.amboinicus. Solubilization of insoluble phosphates illustrate that the isolate can be deployed as a bioinoculants to minimize the fertilizer application. The role of ACC deaminase in decreasing the ethylene levels by the enzymatic hydrolysis of ACC into α-ketobutyric acid and ammonia was reported as one of the major traits of endophytes in promoting root and plant growth. Penrose and Glick [26] reported that greater or equal to 20 nmol α-ketobutyrate mg-1 h-1 ACC production by the isolate is sufficient to promote plant growth.
Siderophore production in endophytic bacteria illustrates iron sequestration mediated pathogen inhibition and plant growth promotion. Siderophore produced by R. globerulus was found to be of trihydroxamate with hexadentate nature.
This study is the first to report the QSI potential of endophytic isolate R. globerulus. Based on the results it can be postulated that secondary metabolites in the cell free lysate of endophytic bacteria degrade HHL and thereby prevents the expression of violacein production. Unfortunately, most of the QSIs available are toxic and unsuitable for human use. Therefore, quorum sensing blockers of probiotic bacteria is an intriguing target for future antimicrobial chemotherapy.
Drenching of P. amboinicus shoot tips with R. globerulus enhanced the number of leaves, shoot and root length with that of untreated plants. Increased herbage yield of O. sanctum on seed treatment with endophytic bacteria in field trials was reported earlier [27]. In the present study, colonization of endophytic bacterium within the leaf tissue of treated plant was confirmed by SEM imaging.
The leaves of P.amboinicus are taken orally to cure different respiratory and intestinal ailments. So, it is inevitable to screen the probiotic potential of endophytic bacteria that resides within the leaves of the plant. Tolerance to different concentration of bile salt and viability at high acidic conditions (pH 2) reflects the probiotic nature of R. globerulus.
Conclusions
Phytochemical constituents of P. amboinicus attribute for its nutraceutical and pharmacological properties. Nonetheless, our research findings confirm that the plant-associated microbiome is a repertoire of metabolic products that contribute for the nutritional and defense pathway of the host plant. QS inhibition by R. globerulus provides a scientific basis for the prevention of pathogen infection in host plant. Probiotic properties of the endophyte confer an added advantage to the therapeutic value of the plant P. amboinicus. Phytochemical constituents and probiotic potential of the endophytic microbiome is a proof of principle for the safety and efficiency of the raw intake of P. amboinicus in ailment prevention. Screening of culture-independent endophytic microbiota of P. amboinicus using high-throughput techniques are in progress.
Acknowledgments
The authors are grateful to Thiagarajar College management. Assistance extended by Gandhigram Rural Institute, Gandhigram for SEM analysis is greatly acknowledged. Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors/editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
Fig. 2 Phylogenetic relationship of bacterial endophyte Rhodococcus globerulus (Accession No: KR013744) and reference bacteria based on 16S rRNA gene sequences (neighbor-joining dendrogram). Maximum parsimony methods are used to construct phylogenetic trees. Bootstrap values are shown near the clades.
Englishhttp://ijcrr.com/abstract.php?article_id=1773http://ijcrr.com/article_html.php?did=1773
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241914EnglishN2017July28HealthcarePrevalence and Gender Differentials of Metabolic Syndrome Among College Students of Kolkata, West Bengal, India
English1418Tanima Paul Das Minati SenEnglish Indranil SahaEnglish Debnath ChaudhuriEnglishAim: The study aims to determine the prevalence of metabolic syndrome (MS) and assess gender differences among college students of Kolkata, West Bengal, India using International Diabetes Federation (IDF) criteria.
Materials and Methods: Design: A cross sectional study was conducted among 397 college going students comprising of 235 females and 162 males aged 18 to 24 yrs from August 2011 to December, 2013. Measurements: Anthropometric measurements were performed by standardized techniques, blood pressure (BP) by sphygmomanometer. Serum glucose, HDL-C and TG were measured in fasting blood. Diagnostic criteria used: MS was identified using IDF criteria.
Results: Prevalence of MS among total population was 4.5%. Significant differences exist between genders with respect to age, BP and HDL-C (p ? 0.05). 9.1% had only central obesity while 12.1% students had any one of the metabolic syndrome components in addition to central obesity.
Conclusion: Many of the college students of Kolkata were having MS or its predisposition. Early identification can be beneficial for planning intervention strategies in college and university settings.
EnglishMetabolic Syndrome, College students, Kolkata, IndiaINTRODUCTION
Metabolic Syndrome (MS) has been defined as clustering of cardiovascular disease risk factors, including hyperinsulinemia / insulin resistance, hypertension, atherogenic dyslipidemia, obesity and glucose intolerance (1-3).The definitions with distinct differences which helps in the interpretation of metabolic syndrome are given by World Health Organization (WHO, 1998); European Group of Insulin Resistance (EGIR); Adult Treatment Panel III (ATPIII, 2001); American Association for Clinical Endocrinologists (AACE,2003), International Diabetes Federation (IDF, 2005) with ethnic specific cut-offs and modified NCEP-ATP III criteria (2). According to the IDF definition, for a person to be defined as having the metabolic syndrome must have central obesity (defined as waist circumference with ethnicity specific values) plus any two of the other four risk factors of MS (4). In adults, MS has been reported to predispose to an increased risk of chronic diseases such as type 2 diabetes mellitus (T2DM) and cardiovascular diseases (CVD) (2), particularly in Asian Indians (5). Prevalence of obesity and MS is rapidly increasing in India and other South Asian countries, leading to increased mortality and morbidity due to CVD and T2DM.About one third of urban South Asians have evidence of MS (6-8).
College students are an understudied population in respect to prevalence of MS. Experts emphasize that MS is becoming increasingly prevalent and parallels the emergent pandemic of overweight and obesity (9) even in young adults (10). Studies document that college students experience weight gain faster than average adults (11-12). Huang et. al. (13) concluded that overweight students are more predisposed to the components of MS. It was also reported that college students make unhealthy food choices and do less physical activities (14).
Therefore, objectives of this study were to find out the prevalence of metabolic syndrome and to assess its pre-disposition among college students of Kolkata, West Bengal, India.
MATERIALS AND METHODS
Study participants
Three hundred ninety seven (397) college students comprising of 235 females and 162 males aged 18 to 24 yrs living in Kolkata were randomly selected from ten colleges for this study. Purpose of the study was explained to them and written consent was obtained. Participation in the study was voluntary and non-remunerative.
Exclusion criteria
Students who were suffering from metabolic abnormalities e.g. Type 2 diabetes mellitus (T2DM) or with a medical history of previous cardiovascular events with prescribed usage of antihypertensive drugs or on hormone therapy (e.g. insulin), pregnant and lactating women, athletes or unwilling participants were excluded from the study.
Ethical considerations
The study protocol has been approved by the Bioethics Committee for Animal and Human Research Studies, University of Calcutta (Ref no. BEHR/1098/2304 dated 22/06/11).
Study Design
Cross sectional study was conducted from August 2011 to December 2013. Participants were recruited via classroom announcements, flyers and word-of-mouth. Survey along with blood sample collection was done randomly in respective institutions.
Anthropometric estimations
Participants were requested to wear light clothes and not to wear shoes/socks for the measurements. Height (nearest 0.5cm), weight (nearest0.1kg), waist (nearest0.2 cm) and hip circumference (nearest 0.2 cm) were obtained using standardized techniques (15). Blood pressure was measured on the right arm of the participants in a relaxed, sitting position with the arm supported at heart level, using a standard mercury sphygmomanometer. All measurements were done in duplicate and the average was recorded (16).
Biochemical estimations
Blood sampling of the students were performed by a trained phlebotomist by venipuncture after10 to 12 hr overnight fast. Samples were transported to the laboratory in an ice bucket within 2hrs of collection and serum was separated by centrifugation of whole blood for 20 minutes at 2000rpm. Fasting blood measures included serum glucose by glucose oxidase – peroxidase method(17), serum total cholesterol (TC) by cholesterol oxidase peroxidase-aminoantipyrine method(18) and serum triglycerides (TG) by glycerol oxidase peroxidase amino antipyrine method (19) using assay kits from Span Diagnostics Limited in a semi autoanalyzer. High density lipoprotein cholesterol (HDL-C) was determined by cholesterol oxidase peroxidase-amino antipyrine method after precipitation of low density lipoprotein cholesterol (LDL-C) and very low density lipoprotein cholesterol (VLDL-C). LDL-C and VLDL-C were calculated using Friedwald’s equation: LDL-C = TC – (HDL-C + TG/5); VLDL-C = TG/5 (18).
Definition and diagnostic criteria used
Metabolic syndrome (MS) was defined according to IDF criteria for MS. According to new IDF definition: for a person to be defined as having MS they must have:: Central obesity (defined as waist circumference ≥ 90 cm for males and ≥ 80 cm for females) as for Asians and any two of the following four factors: raised triglycerides (≥ 150 mg/dl), reduced HDL-C(Englishhttp://ijcrr.com/abstract.php?article_id=1774http://ijcrr.com/article_html.php?did=1774
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241914EnglishN2017July28HealthcareComparative Study of Social Support among Individual, Team and Dual Sports Athletes
English1922Amandeep SinghEnglish Amritpreet SinghEnglishAim: The aim of present study was to compare the social support among individual, team and dual sports athletes.
Methodology: For this purpose, the investigators had selected one hundred eighty (Individual sport=60, Team sport=60, Dual sport=60) male inter-college individual, team and dual sports athletes between the age 17-28 years as subjects. All the subjects were assessed for social support with the help of Social Support Questionnaire given by Zimet et al. (1988).
Results: One-way analysis of variance (ANOVA) revealed that the individual, team and dual sport athletes had significant differences on the sub-variables family (pEnglishSocial Support, Players, Team Sport, Dual Sport, AthletesINTRODUCTION
Sports psychology is the scientific study of mind, emotion, and behavior as it relates to athletic performance and physical activity. The mental demands of rigorous competition can be enormous, making sports psychology a crucial part of any athlete’s training regimen. Psycho-social factors played an important role in the performance and well-being of athletes. Athletes need the positive support of teammates, coaches, parents and friends especially when the athlete feels that he/she is not performing well or realizing his/her potential (Weinberg and Gould, 2003). Social support is a multi-dimensional construct (Udry, 1996) which allows for many possible providers of support as well as various forms. Expressing emotional support, tangible support, informational support and esteem support are all examples of supportive social behaviours (Albrecht and Adelman, 1984).
Social support is often used in a broad sense, including social integration. However, Social integration refers to the structure and quantity of social relationships, such as the size and density of networks and the frequency of interaction, but also sometimes to the subjective perception of embeddedness. There have been recent indications that social support resources play an important role in athlete retention and success (Botterill, 2004). Social support is associated with better psychological health in general and reduces the negative psychological consequences of exposure to stressful life events (Cohen and Wills, 1985). Social support has also been defined as a those social interactions or relationships that individuals with actual assistance or that embed individuals within a social system believed to provide love, caring or sense of attachment to a valued social group (Hobfoll, 1988). Generally, social support refers to knowing that one is loved and cared for and that others will do all they can when a problem arises (Sarason, 1990). Wallston et al. (1983) reported that various sources or types of social support contribute to different outcomes in physical health. In recent years there have been many studies of the relationship among adjustment, social support, and psychological well-being or life satisfaction (Hardy et al, 1991; Malinauskas, 2008; Petrie, 1992, 1993; Ryska and Yin, 1999; Smith et al, 1990).
There have been recent indications that social support resources play an important role in athlete retention and success (Botterill, 2004; Bruner, 2002; Halliwell, 2004). Generally, social support refers to knowing that one is loved and cared for and that others will do all they can when a problem arises (Sarason et al., 1990). Sarason and colleagues concluded that the essence of social support lies in the individuals’ beliefs that they have valued providers who display concern, and are willing to assist in times of need. In sports, social support might influence performance by providing advice about tactics and game plans, or by increasing positive effect, leading to a greater likelihood of experiencing flow states (Cohen, 1988; Rees et al., 1999). Alternatively, social support might buffer the negative impact of stress on performance. Pines et al. (1981) suggested six types of social support offered by providers such as listening, emotional, emotional challenge, shared social reality, technical appreciation, and technical challenge. Rosenfeld and Richman (1997) proposed two additional support types viz. tangible assistance (providing financial assistance) and personal assistance (driving the athlete somewhere).
While considering the paramount importance of psychological variables with regard to sports, the present study aims to analyze and compare the social support in individual, team and dual sports athletes.
METHODOLOGY
For the purpose of the present study, one hundred eighty male inter-college individual, team and dual sports players between the age group of 17-28 years were selected as subjects. The subjects were purposively selected from various colleges of Guru Nanak Dev University, Amritsar. The subjects include 60 individual sports athletes, 60 team sports players and 60 dual sports players. All the subjects, after having been informed about the objective and protocol of the study, gave their consent and volunteered to participate in this study.
Social Support
The social support among the individual, team and dual sport athletes was assessed with the help of Social Support Questionnaire given by Zimet et al. (1988). Multi Dimensional Perceived Social Support is 12 items instrument that measure an individual’s perceived level of social support. It is designed to assess perceptions of social support adequacy from specific sources, family, friends and significant others. Participate were asked to rate their perceived level of social support on a seven Likert scale from 1 very strongly disagree to 7 very strongly agree. The whole scale has 12 items score can range from 1-84 (higher scores indicating a greater level of social support.
It has been found that overall score and the scores for subscale (family, friend and significant others) separately show acceptable published test-retest reliability, factors of validity and construct validity. Significant others and friends factors were found to be more moderately correlates (r= .63), the family subscale was found to be more independence from the other, with correlations of (.24) and (.34) with significant others and friend, respectively. Further reliability ranged from .84 to .92 for the scale as a whole.
Factors of Social Support
Family: family is measured by item 3, 4,8,11. These items are “My family really tries to help me”, “I get the emotional help and support I need from my family”, “I can talk about my problems with my family” and “My family is willing to help me make decisions”.
Friends: friends are measured by item 6, 7,9,12. These items are “My friends really try to help me”, “I can count my friends when things go wrong”, “I have friends with whom I can share my joys and sorrows” and “I can talk about my problems with my friends”.
Other significant persons: other significant persons are measured by items 1, 2,5,10. These items are “There is a special person around when I am in need”, There is a special person with whom I can share my joys and sorrows”, I have a special person who is a real source of comfort to me” and “There is special person in my life that who cares about my feelings”.
Scoring: The present scale is a multiple choice responses presented in a continuous responses range from very strongly disagree, strongly disagree, mildly disagree, neutral, mildly agree, strongly agree to very strongly agree. In this seven point scale, the responses are given weight from 1 to 7 as shown below. Therefore, a higher score reflects better social support.
Statistical Analysis
One Way Analysis of Variance (ANOVA) was employed to compare the level of social support among individual, team and dual sports athletes. Where ‘F’ values were found significant, LSD (Least Significant Difference) Post-hoc test was applied to find out the direction and degree of difference. For testing the hypotheses, the level of significance was set at 0.05.
RESULTS
Table 2 presents the descriptive statistics and F-values of the social support and sub-variables of social support viz. family, friends and other significant persons of the individual, team and dual sport athletes. Table 3 presents the LSD (Least Significant Difference) Post-hoc analysis of sub-variables of social support among individual, team and dual sport athletes. The results revealed that there were significant differences (F=10.74, p=0.00) among the individual, team and dual sport athletes on the sub-variable family. Team sport athletes were found to have highest mean value on sub-variable family followed by dual and individual sport athletes respectively. The LSD post-hoc analysis revealed that the team sport athletes were significantly better than the dual (pEnglishhttp://ijcrr.com/abstract.php?article_id=1775http://ijcrr.com/article_html.php?did=1775
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241914EnglishN2017July28TechnologySoft Computing Techniques in Mobile Adhoc network : A Technical Overview
English2325V. PreethaEnglish K. ChitraEnglishMobile ad-hoc network is widely used in various communications due to its easier deployment and cost effectiveness. In recent technological advancement, Manet is used in all types of environment including pervasive computing, Internet of Things, military applications, Disaster recoveries, Vehicle tracking etc. In Manets, the research is going on in the field of Routing such as Hierarchical routing, Zone based routing, Distance based routing etc. Manets in case of sensitive networks has to face the challenge of security and in many cases the existing protocol is enhanced with security using cryptographic techniques. In recent researches, Soft computing techniques like Genetic Algorithm, Fuzzy logic, Neural Networks are employed in Manets in every area of research. We propose an attempt to survey the applications of soft computing techniques in Manet and in particular Artificial Neural Network learning technique. Since Soft computing techniques are efficient for solving complex problems, our focus is towards this survey approach in Manets.
EnglishSoft computing techniques, Genetic algorithm, Fuzzy logic, Neural network, Mobile adhoc networkINTRODUCTION
Soft computing is a new disciplinary for Artificial Intelligence which induces inductive reasoning for Intelligent systems and for automated systems. It is based on Human behavior models to emulate the human mind as closely as possible. Unlike Hard computing, Soft computing deals with approximate models. Evolutionary computation is a subfield of Artificial Intelligence which involves combinatorial optimization problems. It uses iterative progress and is a class of global search paradigm by generating finite state automata. Genetic Algorithm was one of the evolutionary computing metaheuristic optimization technique. Particle swarm optimization is one of the evolutionary computation technique. Fuzzy logic is another soft computing technique. It is used as a problem solving methodology from small microcontrollers to large scale control systems. It can be used both in hardware and software. Neural Computing is an important new concept which combines the other areas such as Neurology and Psychology. Many Scientists believe that conscious Neural Network is realistic Possibility.[1]. Neural Network is widely used in Forecasting, classification problems. Hybrid systems can be classified as Neuro fuzzy hybrid system, Neuron genetic hybrid system and Fuzzy genetic hybrid system. The Hybrid systems combines the Partnerships of any two methodologies for problem solving.
SOFT COMPUTING TECHNIQUES IN MANET
Mobile Adhoc network has to face many challenges for effective communication. Due to varying topology, Routing in these networks is very complex .Different types of Routing techniques have been adopted by focusing on shortest stable path. Mobility Prediction, Link Expiration time will be helpful for predicting the future of the nodes. As a result many strategies such as Mobility Management, Location Management are considered in MANETS. Selecting the Optimal Path in Routing is an important factor. While considering the Hierarchical routing, maintaining the stability of clusters is a challenging issue. The aim of good routing to achieve the better Quality of service. R. K. Ghosh et.al considered three central problems such as base station placement, channel assignment and mobility management [2] in their survey. By considering the challenges in Manet, soft Computing Techniques will make an attempt to solve the major challenges to some extent. The use of soft computing techniques in Manets is tabulated as :
ARTIFICIAL NEURAL NETWORK
Artificial neural networks (ANNs) has led to a remarkable pitch in mobile adhoc network research. Artificial Neural Networks are very efficient for supervised and unsupervised learning and reduces the complexity of the problems by prior training but with some uncertainty. The term Neural Network will refer to a circuit of biological neurons. Like Human brain, the neurons will perform the physiological function. ANNs has been used as an alternative to non linear regression and cluster analysis for statistical analysis and data modeling. The main characteristic of ANN is their ability to learn. The learning is performed by adjusting the synaptic weights and the input stimuli and the output response are studied. The adaptation process is learned with the desired output. Neural Network has been categorized as supervised learning network and unsupervised learning network. Perceptron Networks, Adaptive linear Neuron, Multiple Adaptive Linear Neuron, Back Propagation Network, Radial Basis Functions Network, Time delay, Wavelet Neural Network fall under supervised Neural Network. Kohonen self organizing map, Fixed weight competitive nets, Adaptive Resonance Theory Network fall under Unsupervised Learning Network. A feed forward neural network is used for prediction with three layers as input layer, Hidden layer and output layer. The back propagation is the learning method widely used. The weight is adjusted in each node in such a way that the error between the desired output and actual output is reduced. To minimize the mean square Error, Gradient descent method was used. To start the training the initial weights are chosen randomly and then the training begins. The back propagation algorithm looks for the minimum Error. Hopfield network approach mainly focuses on the energy function containing the objective function. Many Researchers have employed many methods of Artificial Neural Network in Manets but we have listed out only the major work related with ANNS. The remaining part of this paper presents state-of-the-art survey of ANN applications in Mobile adhoc networks. The list of authors and their work in Manet using Artificial Neural Network.
Heni Kaaniche et.al. used Neural network for mobility prediction and identified stable paths to improve routing by reducing the overhead and the number of connection interruptions. They employed the multilayer and recurrent neural network using back propagation through time algorithm for training. Jyoti Prakash Singh et.al. have tried to evaluate the applicability and capability of artificial neural network for prediction of end-to-end packet delay in mobile ad hoc network environment. They developed two models on Radial Basis Function (RBF) network and Generalized Regression Neural Network (GRNN). S. Gangwar et.al used the ART network for cluster head selection in Manet. ART1, is an unsupervised learning technique. Manisha et.al. provided enhanced security to Manet using the Back Propagation Method of Artificial Neural Networks and eliminated the use of files for storing the password. ANNS is applied in every area of Manet research especially in the case of future prediction.
CONCLUSION
Manets are facing Complex multiobjective optimization Problems which can be solved by Soft computing Techniques. This paper focuses on the challenges in Manet and listed out the Authors work using soft computing Techniques in Manet. The remaining paper surveyed the different types of Neural Network and the authors work related with Neural Network in Manet. Thus advantages of using soft computing includes prediction, problem solving, and robustness. This survey highlighted the soft computing technique employed in specific research area of MANET
Englishhttp://ijcrr.com/abstract.php?article_id=1776http://ijcrr.com/article_html.php?did=1776
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Heni Kaaniche and Farouk Kamoun,” Mobility Prediction in Wireless Ad Hoc Networks using Neural Network”, Journal of Telecommunications,Vol.2. Issue 1,(April 2010).
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Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-5241914EnglishN2017July28TechnologySchumann Resonance Mode Variation during Seismic Activity: A Review
English2630Pranab HazraEnglish Shreya ChatterjeeEnglish Moumita BanerjeeEnglishSeismic activity and thunderstorm centers have strong relation because of which fourth mode (26Hz) of Schumann Resonance (SR) is varying. SR has numbers of resonance frequencies like first mode (8 Hz), second mode (14 Hz), third mode (20 Hz) and fourth mode (26 Hz). The variation of 4th mode of SR is taken into account due to the seismic effect. Characteristics of ionosphere changes on surface during and aftershock the main shock (https://www.britannica.com/science/aftershock-geology). The effective height of the ionosphere lower boundary is reduced to a few tens of kilometer. The lower ionosphere and mesosphere is modified by the seismic source detection of modification in higher frequency band. The Schumann Resonance signal is supported by electromagnetic radiation from the global thunderstorms and the anomaly in SR fourth mode frequency i.e. the shifting of frequency due to lowering of Ionosphere height is accumulated in this paper.
EnglishSchumann resonance, Earthquake, ELF, Fourth mode SR, Ionosphere, ThunderstormINTRODUCTION
Seismic Activity
The displacement of two or more plates causes faults and resulting shaking in the earth surface and energy released in the form of earthquake. The volcanic or magmatic activity may also be associated with ground shaking. If any of two blocks of the earth suddenly slip past one another then it happens. The fig.1 shows surface slip due to which fault plane created. In the location where the earthquake starts below earth’s surface is called the hypocenter, and the location which directly above on surface of the earth is called the epicenter.
Sometimes some smaller earthquakes are happening in the same place where the larger earthquake follows that kind of earthquake has foreshocks. During the strong seismo-ionospheric coupling processes in the earthquake preparation zone, underground gas discharges carry submicron aerosols with them which enhance the intensity of electric field at the near ground due to the drop in air conductivity created by aerosols. Seismo-electromagnetic emissions have been observed at low frequency bands in the seismically active zones prior to the incidence of any large earthquake (Nagao et al., 2002) which are different from lightning induced and technogenic emissions. On the event of strong earthquake, the near ground of the atmospheric layer becomes ionized and generates electric field which introduces particle acceleration thereby exciting local plasma instabilities.
The tectonic plates are moving continually and get stuck at the edges by the friction. Earthquake releases energy in the form of waves that travel through the earth's crust when the stress on the edge overcomes the friction. Due to the sudden release of energy in the Earth's crust it creates earthquakes.
Schumann resonances (SR) Phenomena
The extremely low frequency (ELF) portion of the Earth's electromagnetic field spectrum which set of spectrum peaks are called Schumann Resonances (SR) ( Fig. 4). Schumann resonances are global electromagnetic resonances is excited by lightning discharges in the cavity formed by the earth-surface and the ionosphere (as shown in fig. 3). The space between surface of the Earth and the conductive ionosphere acts as a closed waveguide for Schumann resonances occur. This waveguide to act as a resonant cavity for electromagnetic waves in the ELF band which result the limited dimensions of the Earth cause. The Schumann Resonances are quasi standing wave electromagnetic waves which exist in this cavity. The several frequencies between 6 and 50 cycles per second; specifically 7.8,14, 20, 26, 33, 39 and 45 Hz, as daily variation of about +/- 0.5 Hz for occur by them. The Earth's ionosphere changes in response to the 11-year cycle of solar activity which is some change due to solar sunspot cycle (fig.2) . They are most easily seen between 2000 and 2200 UT.
Global lightning was used to monitor global lightning activity about 2000 thunderstorms around the globe given from Schumann resonance studies. The background Schumann resonance signal is directly linked from producing ~50 lightning events per second, thunderstorms. Schumann resonance records are a complex problem to the spatial lightning distribution: the lightning intensity estimate in order .The distance from lightning sources and the wave propagation between the source and the observer both are the necessary account of its . The cavity is spherically symmetric assumes of this technique. Therefore believed to affect the resonance that does not include known cavity asymmetries propagation properties of electromagnetic waves in the system.
Diurnal variations meant by the background Schumann resonance power spectrum. Global lightning activity and the state of the Earth–ionosphere cavity between the source region and the observer both are the properties of characteristic Schumann resonance diurnal record reflects. The vertical electric field is independent of the direction of the source relative to the observer which is a measure of global lightning.
The Schumann resonances transients (Q bursts) and the transient luminous events (TLEs) both are related. In 1995 Boccippio et al that sprites, the most common TLE, region of a thunderstorm system to the produced by positive cloud-to-ground lightning occurring in the strait which are accompanied by Q-burst in the Schumann resonances band. The occurrences of sprites are revealing recent observations .Q bursts are highly correlated and Schumann resonances data can possibly be used to estimate the global occurrence rate of sprites.
The global temperature and the Schumann resonances are also monitored. There are increases nonlinearly with temperature between Schumann resonance and temperature is lightning flash rate. The nonlinearity of the lightning-to-temperature relation provides a natural amplifier of the temperature changes .It makes Schumann resonance a sensitive "thermometer". The global mean temperature in the low latitude range for only Hew component (correlation coefficient about -0.9) which correlate well Extracted semiannual variations of the SR intensity. Two horizontal magnetic components are negative relation with the global thunderstorm activity. A significant correlation between the middle latitude temperature and the annual component of SR energy until the semi-annual component of the SR energy and low latitude temperature both are also significant correlation
Tropospheric water vapor is a element direct effects as a greenhouse gas. global lightning activity and upper-tropospheric water-vapour variability both are closely linked, upper-tropospheric water-vapour changes can be inferred from records of global lightning activity for suggesting of a single location on the Earth's surface observations to any obtain. The thunderstorms transport large amounts of water vapour into the upper troposphere and thereby dominate the variations of global upper-tropospheric water vapour while producing most of the lightning on Earth that correlation reflects the fact of continental deep-convective. Observed easily, that global lightning induces Schumann resonances, an electromagnetic phenomenon are in the atmosphere
Review of Literature
Third mode to fourth mode of Schumann Resonance is taken into consideration because of shifting of frequency in time frame of large earthquake. Earth’s surface and lower ionosphere made a wave guide. The ionization occurred at about 50 Km above by solar radiation and cosmic rays. Which ionization is completely different at day time from night time [Plok 1982].The conductivity of SR signal changes as the lower boundary changes. Here the lower band of the ionosphere acts as the earth-ionosphere cavity controller. The observed variations were explained by the variations in the source-receiver geometry [Balser and Wagner, 1960], and it was concluded that no particular systematic changes of the ionosphere are needed to explain these variations [Madden and Thompson, 1965].
SR Anomaly during Large Earthquake:
The two waves, direct wave generated from the active thunderstorm center and scattered wave from epicentral region are interfere which results the frequency shifting of higher mode of SR. In this wave the magnetic field component propagating in north-south meridian plane [Haykawa et al., 2005].In some model it was assumed that the conductivity increased due to Total Electron Content (TEC) enhancement in epicentral region. During thunderstroms electromagnetic wave generated by the excitation of lightning [S.S De et al., 2008]. Some relation also found the power of the 1st mode of SR and the global temperature variations. Aerosol particles moves randomly in the vicinity of the earthquake zone and as because of the connective nature of atmosphere there is a directional flow is also there which causes the atmospheric temperature changes. The movement of electromagnetic wave also affected within the earth-ionosphere cavity which in turn changes in SR waves.
The Stratton-chu integral equation technique when constructing the electromagnetic field is non-uniform earth ionosphere cavity [Nickolaenko el. al.2002].The vertical electric field in the earth ionosphere cavity with a localized non- uniform is a combination of direct and scattered waves:
Er = E1 + E2
E1 is the primary wave, the 2nd wave E2 assuming that the seismic activity locally modifies the ionosphere height.
For Taiwan earthquake the records were done at Nakatsugawa observatory where narrowband transient waves observed in SR fourth mode [Ohta et. al.,2001,2003]. The narrow band continuous signal in the horizontal magnetic field component increases the fourth mode Schumann Resonance by two times before and after the main shock [Nicolaenko et al.,2005]. The large earthquake (M=7.8) occurred in Taiwan on 26 December 2006, the ELF signals were recorded at Moshiri (44.29ºN, 142.21ºE) in Hokkaido, Japan, which indicates the anomalous behavior in the Schumann resonance mode, ELF radio wave caused by the lower ionospheric depression around the earthquake epicenter [Hayakawa et al.,2008].
The non-uniformity of the scattered waves is due to the inhomogeneity of the ground surface, irregular conductivity of atmosphere. An isotropic nature of ionosphere is considered for the direct wave [Molchanov et al., 1995].The spatial distribution of ULF magnetic and electric field within the scattered wave are responsible for intensity enhancement of SR and this spatial distribution is effective for 100-200Km horizontal distances [Molchanov et al., 1995] i.e. the source and observatory distance should be within the range of spatial distribution for observing the changes in SR.
Discussion
The shifting of frequencies in fourth mode (26 Hz) and first mode (8Hz) of SR are highlighted in this paper. Two to three days before the main shock the height of the lower ionosphere reduced by near about 20Km which turns into some modification in the ELF electric field. This modification may cause the shift in peak frequencies by certain small percent [Nickolaenko and Hayakawa, 2002]. So SR is considered to a review of lower ionosphere [Hayakawa, 2007; Molchanov and Hayakawa, 2008]. East-West component (BEW) and North-South component (BNS) of the horizontal electrified increases and the vertical component Ez also increased in fourth mode of Schumann resonance. The main responsible is BEW component compare to BNS component.
Three global lightning centers situated in America, Africa, and Asia as shown in fig. 5 indicated by ‘S’. These three centers are responsible for contributing the SR signals by the generated lightning strokes at around Universal Time (8±1) h for Asia, (15±1)h for Africa and (21±1)h for America. The observatory and the epicenter of the earthquake are different and so the radio signals (ELF) reached to the observer in different direction in a day [Nickolaenko and Hayakawa, 2002]. In -Moshiri (Japan) earthquake in Hokkaido, on 26 December 2006, the modification due to the lowering of ionosphere occurred in the interval of 21:00 UT±1 h which was contributed by the American thunderstorm center and irregular behavior observed on 23 December 2006.
It was observed (fig. 6) the ELF signal perturbed due to the lowering of the lower ionosphere height around the epicentral region of earthquake. In case of Taiwan earthquake in September 1999 the Q-factor for 4th mode of SR becomes nearly 10 which is approximately double the natural SR value because of introduction of some train noise [Nickolaenko and Hayakawa 2002]. In this case it was strongly established that this anomaly as because of thunderstorm center at America. From the study of behavior of several earthquake mainly in Japan and Taiwan, it is observed that the fourth mode Schumann Resonance shows the anomaly for the larger earthquake having magnitude of greater than or equal to 6.
Conclusion
The radio ELF signal generated from the thunderstorm centers known as direct wave are enter in to the earth-ionosphere cavity and propagate within the cavity but because of the depression of lower ionosphere the propagation takes place differently. Scattered wave, generated from the disturbed region above the earthquake epicenter has different path length with respect to direct wave. These two waves make interference in SR modes which is responsible for shifting of Schumann Resonance mode by nearly 1Hz almost 2-3 days before the large shock (Hayakawa et al., 2008). Variation of Schumann Resonance also depends on source and observer distance. (https://www.revolvy.com/topic/Schumann%20resonance and item_type=to pic). An anomaly in the SR (Fourth Mode) signals depends on the magnitude of earthquake and the distance between observatory and earthquake epicenter. For larger earthquake, SR frequency shift is higher than the small (not less than M=6) earthquake. Convective nature of the atmosphere changes the aerosol movement and the temporal changes may occur which contribute to the SR modes which may also be taken into consideration.
Acknowledgements: The authors acknowledge with thanks the support from Narula Institute of Technology, Agarpara, Kolkata, India for encouraging in research work and giving scope in this regard. We also thankful to the department of Electronics and Communication Engineering of Narula Institute of Technology, for carrying out the study.
Authors acknowledge the immense help received from the scholars whose articles are cited and included in references of this manuscript. The authors are also grateful to authors / editors / publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed.
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