Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524199EnglishN2017May15General SciencesA correlative Study between the Implementation of Rhythmic System and the Hieroglyphics Substantial English0105Amit ChauhanEnglish Aditi ChauhanEnglish Jyoti SinghEnglish S. K. ShuklaEnglishAim: The aim of the study is to find out the correlation between the hieroglyphics substantial and the rhythmic system of an individual. Methodology: In this study, we analyzed the three aspects of handwriting such as the legitimate writing, forged and the disguise handwriting samples which were taken to conclude the positive identity of the suspect by the implementation of rhythmic system and adoption of few parameters. Results: The perfection of similarity is not possible in human work, so some variations were noticed up to a range of ±0.2cm in the measurement while the angle variation was noticed up to a level of 1.5?, while the fourth component was noticed highly significant that is 0.1 cm variation in the handwriting samples. Conclusion: When any kind of substantial is forged by adopting any kind of practice with perfection though the elements of interpersonal and interpretational variations comes in. This study signifies that adopted parameters gives the concluding identification of an individual and could be endorse by the investigator and could be put on par of all other methods used for questioned documents examination. EnglishHandwritten material, Forgery, Metric, Identification, Investigation, etcINTRODUCTION At the time of advancement, when technology and scientific methods have taken over the security system and for the safety of society. The identity of individuals is converted into computational language; written materials have been obtained inform of the photocopies or print out from the different systems. Yet, we rely on the documents which are usually kept for records with the signatures of the authority. These documents which consist of any kind of information either visible or invisible that ultimately conveys message to another person[1, 2]. Not in our communal society, even criminals have also not been slow to realize the value of document in one’s life as how they can impact person’s possession and wealth. This trend of artifice often comes in form of the disputed documents, signatures, forgery[3,4]. The origins of the documents which are under dispute are known as questioned document and required a documents examiner to prove their authenticity.  Alteration and edition in security featured documents such as bank cheque’s, willpapers, power of attorney are very common and in the trend since ages. Doing alteration in signatures and writings are becoming easier for forger on the contrary examining them is difficult. Hence handwriting expert is required who has intensive experience and training in his field. Since, the science of handwriting is based on the premise that now two individuals can reproduce the same writing even their own writing and encountered in form of variation[5,6,7]. Variations are natural deviations that occur in a person’s handwriting and known as a complex perceptual motor task. Every individual possess characteristics of their own handwriting into two subdivisions such class characteristics and individual characteristics such as master patterns are unique to individual and helps in identifying the suspects[8].When an individual modifies his/ her own handwriting willfully to conceal his identity is known as disguise handwriting, in which the author tries to keep his identity anonymous and this is mostly used during comparison of specimen[9,10]. The suspect usually change the shape, style of the letter formation, they adopt the someone else’s countenance to write so that no one can find the similarities in first appearance between them. In the process of forgery, the suspect imitate and adapts the writing features of someone else’s with the intent to create a fraudulent document.    In this process, an instinctive process of adding disguise is to change this instinctive process takes a significant and cognizant effort on the part of the author’s to assess a variety of line quality features. In some cases, where the suspects are known encountered and familiar with the exact countenance of victim, the recognition become a tenacious task for examiner to arbitrate their identity[11,12]. As we all know that variations are the intrical part of the handwriting which may not be in the form of countenance, but it could be encountered in form of the metric of suspects written characteristics. The thing that will remain same in one’s handwriting is the measurement of letters and words, which are left unknowingly by the forger. If a person disguises his handwriting he won’t be able to exclude his metric features which are deeply rooted in the handwriting in form of the length, angle from the line of writing and height of the peculiar letters and words will remain same. For, this purpose three types of the handwriting samples were collected which includes, genuine handwriting, forgery, and the disguise handwriting substantial[13,14]. In this study, we focused on four parameters namely, length of specific word, Angle from the line of writing following by the Eudidean geometry’s theorem, height of the two highest similar letters present in word, and the height of anyone small letter present in word to examine the occurrence of the variation. The results of the study were dreadful that even the forger try to avoid their writing style and formation of letters followed by the change in the individual characteristics even left their identity in form of metric of written substantial. Hence, by adopting the rhythmic system in the examination of hieroglyphics substantial can proved an advance step towards conclusive identification. This process can be put on the par of the all questioned documents examination process in court of law[15]. METHODOLOGY Since diversified types of document examination process has been carried out and some of them still has to come. Questioned document examination is very important aspect for the purpose of identification of suspect in forensic investigation and to establish their conclusive identification with absolute certainty. Hence, sometimes the variations between the interpersonal and interpretational comes in a form of apprehension, therefore to establish the conclusive identification of the author, we implement the metric system to identify the suspect. According to “EUDIDEAN GEOMETRY “there is a single infinity location where all lines meet. All lines intersect at infinity and a whole collection of additional points in one common direction provide projective geometry. MATERIAL For this pilot study, we collected 25 samples including males and females from the population of national capital region of India. The sample selection was done randomly without adopting any specific criteria or age group. The subjects were informed prior to sampling process and ethical considerations were taken. Each subject was requested to provide 3 samples in different forms such as genuine handwriting, disguised and forgery in respect of an available printed sentence. METHODS For the sampling purpose, all the samples were collected with the help of blue/black/green pen on white A 4 size sheet. All the samples were preserved in a white paper envelop to avoid any kind of moisture effect or ingredient or flow of ink. For the analysis of the sample pencil, scale, protector, hand lens of 5x were used. All the samples are photographed with the help of… For the statistical purpose SPSS software were used and significance level was examined. HYPOTHESIS In the present study, it was considered that the description of an individual can be constituted and a hypothesis was formulated. According to that- H0signifies that implementation of rhythmic system has concluded the constitute of suspect at 95% level of confidence. While, H1 Implementation of metric system has not a higher significance level at 95% of confidence level. RESULT In time, the handwriting/signatures have become highly individualized often in a shape style which distinct it from the original handwriting of the author/suspect. Sometime it depends upon the combined effect, condition, health issues and situation of the author in which he/she writes. The signatures assume greater skills; movement becomes less primitive and elementary and the elements approximate to a greater or lesser degree of the writing that individual studies with more advanced authors inherits. The conditions under which these standards are prepared make it imperative that certain precautions be observed so that their comparison value is not impaired. In many instances the person who prepared the writing will freely admit the fact, especially when it in no way seems to incriminate him or to prejudice his case. Stipulations about the genuineness of certain standards can often be obtained if asked for, To begin identification, the known writing is examined for naturalness in execution and for repeated consistent features that determine the established habit of the writer [16]. Some examiners begin an examination by formulating the hypothesis that the questioned writing is not written by the writer of the exemplar writing and attempt to prove the hypothesis. If the hypothesis cannot be proven based on the evidence, then the examiner can begin again using the hypothesis that the same person wrote both the questioned and exemplar writings[17]. In this study, the four parameters namely length of the word from starting pen pause to the end pen pause, height of the word at the highest letters from the line of the writing, angle of the highest point (letter) from the line of the writing, and the length of the letter present in between the word, were presumed for the identification establishment (figure-1). In genuine writing of the author, the measurement of the all three words (as described in table no. 1) for “count” 1.5cm., For chicken 2.3cm, and for hatched 1.9 cm. While in disguise handwriting, it was; for count 1.9 cm., for chicken 2.6cm. and for hatched 2.5cm. For the same author, the measurements in forgery of count was 1.9cm, for word chicken 2.9 cm., and for hatched it was 2.3 cm. In which the mean of word count in all three parameters; values lie in between 1.77 cm, S.D. value as 0.03 and SEM was 0.13 which is an indication of natural variation.   For second component of first parameter, the values lie in between 2.6 cm, S.D. value as 0.3 and SEM was 0.17. For third component of first parameter, the values lie in between 2.2 cm, S.D. value as 0.3 and SEM was 0.17. For the second parameter, height of word at two highest letters (t) in word “count” in all three aspects from line of writing in writing was 0.35 cm, 0.30 and 0.50 cm, while for word “chicken” in handwriting, it was 0.45cm, 0.40cm and 0.70cm., and for third word “hatched” it was 0.40cm, 0.45cm & 0.50cm. In which the mean values lie in between 5.83 cm, S.D. value as 1.08 and SEM was 0.57 which is an indication of natural variation. Similar like of it, in second word observed values lie in between 8 cm, S.D. value as 1 and SEM was 0.76 and in third values lie in between 6.5 cm, S.D. value as 1.75 and SEM was 0.76. The third parameter, Angle of highest point of letter in the word “count” from the line of writing, in all three handwriting was noticed up to 5?, 5.5?, and 7 ?. And the mean was 5.83, S.D was 1.08 while the standard error mean (0.57) was noticed. While for second letter “chicken” the measurement of hand writing angles from line of writing were 8?, 9 ?, 7?, whose mean value was 8cm, S.D. 1cm and SEM was 0.76cm were observed. In the last third word “Count” the measurements of angles in all three aspects from the line of writing were obtained as7 ?, 7.5?, 5 ?. In which the mean values lie in between 6.5 cm, S.D. value as 1.75 and SEM was 0.76 which is an indication of natural variation. At the duration of sample examination, which was considered the fourth parameter in the handwritten substantial; the height of the middle letters “c” present in the all three words was also noticed whose dimensions were 0.30 cm, 0.35 cm and 0.30 cm. In which the mean values lie in between 0.31 cm, S.D. value as 0.0008 and SEM was 0.01 which is an indication of natural variation. Similar another measurement is mentioned are shown in table 2 below- In this study, it was noticed that there is a minor change in the measurement of the different handwritten material of same individual. If a person knowingly make changes in his/her writing, in that case various letters formations are noticed because the person remain alert. While imposed the same measurements of their own handwriting which was identical for individuality. DISCUSSION As per the statement mentioned in methodology, the measurements were taken from the genuine handwriting, disguise and forgery and three words (count, chicken, hatched) were selected in each type of handwritten samples. Since it is known that human is not a machine who will work similar like machine so, variation was noticed up to a range of ±0.2cm in the measurement while the angle variation was noticed up to a level of 1.5?, while the fourth component was noticed highly significant that is 0.1 cm variation in the handwriting samples. Some of the variations may be the presence of human error during examination or instruments variation. These parameters can be adopted for the examination of handwritten materials when the identity of suspect become a necessity and other examination fail to provide the clue. Such examination of substantial will prove their importance in getting to nab the suspect and can be put on the par of other exists techniques of examination in court of law. CONCLUSION In this present study, an attempt has been made for the identification of the suspect with the implementation of rhythmic system from the questioned handwriting substantial. As we have contemplated that an imposture may be transpired in diverse ways. For one of the successful to the expansion that it prevents recognition under concealed by the expert study, it must duplicate all the attributes of the authentic signatures to a degree that disparities are few and can be attributed to chance or to the normal range of variation in genuine substantial. Not only the form of letters but the qualities of writing movement must be accurately reproduced so that the problem involves imitating simultaneously a few characteristics rather than each one singly.  There are curbing factors that sometimes preclude absolute identification and eliminations of forger. The outcomes of this study are very satisfactory and encouraging that will be helpful in the examination of different type of handwritten substantial and facilitate with a new approach towards the examination. The inquisitor should be aware about the limitation of some type of evidences and shouldn’t be inveigled by the circumstances of the case and render any subjective conclusion. There is numerous mechanism of document examination which allows the litigators to testify in court of law. Questioned substantial exploration is a challenging and distinct area of research that mainly deals with new investigative techniques and with a new expertise in the area of examination. In this area of interest, the researchers become comprised further to enlarge the area which may require circumspect continued education keeping abreast of new evolution and technology. These types of advancement can carry forward the examination of document for establishing the identity and can put on the par above of all evidences in court of law. ACKNOWLEDGEMENT The Authors acknowledge the immense help received from the Honorable Founder President of Amity University and to the Chancellor who provide all the facilities in the university for this work. The authors are also grateful to editors/publishers of all those articles, journals and books from where the literature for this article has been reviewed and discussed. Conflict of Interest; Nil Source of Funding; Nil         Englishhttp://ijcrr.com/abstract.php?article_id=811http://ijcrr.com/article_html.php?did=811 Kam M, Gummadidala K, Fielding G, Conn R. Signature authentication by forensic document examiners. J Forensic Sci 2001 vol. 46 Pp; 884–888. Huber RA, Headrick AM. Handwriting Identification: Facts and Fundamentals. Boca Raton, FL, CRC Press, 1999, p. 29.  Homewood SL, Oleksow DL, Leaver WL. Questioned Document Evidence. California Lawyers 1998: VIII-4. Hilton O. Scientific Examination of Documents. Chicago: Callaghan & Company, 1956. Srihari SN, Cha S-H, Arora H, Lee S. Individuality of handwriting. J Forensic Sci 2002, Vol. 47, Pp ;856–872. Mehta MK. Identification of handwriting and cross examination of experts. Fourth edition, 1970. Ron N. Morris. Forensic Handwriting Identification: Fundamental Concepts and Principles”, Academic Press, Tokyo 2000 Ordway. Hilton. Scientific Examination of Questioned Documents.  Elsevier Science Publishers B.V. 1982 Billy Prior Bates. Identification System Of Questioned Documents (ISQD). Charles C Thomas Publishers 1970. Willson. R. Harrison (1958), “Suspect Documents- Their Scientific Examination”, Nelson- Hall Inc. Canal St., Chicago, IL. Russell. A. Gregory. Identification of Disputed Documents, Fingerprints and Ballistics. Fifth Edition, Eastern Book Company. 2005 Alford, E.F. Disguised Handwriting. A Statistical Survey of How Handwriting is Most Frequently Disguised. Journal of Forensic Sciences, 15 (4) 1970, October, pp.476-488 Alford, E.F. and Dick, R.M. Intentional Disguise in Court-ordered Handwriting Specimens. Journal of Police Science and Administration, Vol. 6 (4) 1978, pp.419-423. Authentication of Disputed Writings by Comparison: The Expert Witness University of Pennsylvania Law Review, Vol. 104 (5) 1956, March, pp.664-678 Baxter, P.G.  Classification and Measurement in Forensic Handwriting Comparisons. Medicine Science & The Law, 13 (1973), pp.166-184. Bethlehem, J. Applied Survey Methods: A Statistical Perspective. New Jersey: John Wiley & Sons, Inc. 2009. Conway, J.V.P. The Identification of Handprinting. The Journal of Criminal Law, Criminology, and Police Science, Vol. 45 (5) 1955, January - February, Pp.605-612.  

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524199EnglishN2017May15General SciencesElectrical properties of Co-Zn ferrite Doped with Silicon English0608S. S. ShindeEnglishFerrites of the composition Co0.7+x Zn0.3 Six Fe2-2x O4 (x = 0.0, 0.1, 0.15, 0.2, 0.25, 3, 4) are prepared by double sintering ceramic method. X-ray diffraction patterns of all the samples show cubic spinal phase. By comparing the calculated and observed X-ray intensity ratios I220/I400 and I400/ I422 and using site preference of various ions, the cation distribution of the present system is determined. At lower concentration of Si up to x = 0.15, the dc resistively (ρdc) increases and for x > 0.15, ρdc is found to decrease. With increase in temperature, dc resistively decreases except for higher concentration of Si (x = 0.3 and 0.4) which show increase in resistivity at lower temperature. Activation energy values show one to one correspondence with resistivity values. Curie temperature is found to decrease with increase in Si concentration. EnglishLattice Parameter, D.C. Resistivity, Activation EnergyINTRODUCTION Studies of spinel ferrites are highly relevant to modern technologies. So that the synthesis of ferrites have become an important part of modern ceramic research. Cobalt-Zinc ferrite is one of the soft ferrites used in electronic devices such as transformer core, electric motors and generators. It is observed that incorporation of diamagnetic, tetravalent and pentavalent ions in the ferrites sufficiently improves their electrical properties. To increase the resistivity and develop the desired microstructure, tetravalent ions like Ti, Sn and Si are substituted in mixed ferrites. It is observed that the resistivity increases with increase in Si4+ content in Ni-Cd ferrite [1]. The addition of tetravalent ions like Ti and Si in Co-Cd mixed ferrite have been studied to understand the role of these ions on the physical properties of ferrites [2]. In our previous study we have reported electrical and dielectric properties of Si substituted Co ferrite [3]. As an extension of this work, present paper reports the effect of Si4+ substitution on electrical properties of Co-Zn mixed ferrite. MATERIAL AND METHODS The ferrites with chemical formula Co0.7+xZn0.3Six Fe2-2xO4 (Where x = 0.0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4) were prepared by standard double sintering ceramic technique. AR grade oxides Fe2O3, CoO, ZnO and SiO2 were thoroughly mixed in required proportions. The samples were then presintered at 7000c for 12 hr in air medium and slowly cooled to room temperature. The presintered powder was milled and sieved to a fine powder. Pellets were prepared by subjecting a small quantity of powder to a pressure of about 12 x 106 kg/m2 by keeping it in a die of diameter 1cm and these pellets were finally sintered at 10500C for about 24 hr in air medium. Cooling to room temperature was carried out at the rate of 800c per hr. The structural and physical properties of the prepared samples were determined by X-ray powder diffractometry technique. The X-ray diffraction patterns were recorded on a diffractometer  PW710 using Cu-K radiation of wavelength 1.54060 A0 in the range of 2q = 100 to 900 and scanning rate of 10 per minute. The temperature and composition dependence of dc resistively (rdc) of all samples was studied by two probe method. For good electrical ohmic contact the pellets were polished and silver pasted on both the surfaces. RESULTS The room temperature X-ray diffraction patterns of all the samples of the present series confirm the single phase formation of spinel. Typical X-ray diffraction patterns of the samples with Si concentration x = 0.2 and x = 0.4 are shown in Figure.1. The values of lattice constant ‘a’ listed in Table 1 show slow decrease with increasing Si content from x = 0.1 to 0.4. According to site preference clergies given by Miller [5], Fe3+ ions get statistically distributed over B-site and A-site, Co2+ has a strong preference on B-site and Zn2+ has strong preference for A-site. Also it is reported that Si4+ ions have strong preference to A-site [6]. X-ray intensities were calculated using the formula suggested by Buerger [7]                                                Ihkl   = | Fhkl  |2  P Lp where notations have their usual meanings. By comparing the calculated and observed X-ray intensity ratios I220/I400 and I400/ I422 and using the above stated site preference of various ions, the cation distribution of the present system can be written as                  For x = 0.0  (Zn0.3 Fe0.7 ) [Co0.7 Fe1.2]                  For x = 0.1 to 0.4 (Co0.05 Zn0.3 Si0.05+y Fe0.6-y) [Co0.75+y  Si0.05Fe1.2-y] Where  x = 0.1 + y, y = 0.0, 0.05, 0.1, 0.15, 0.2, 0.3 The values of dc resistivity rdc obtained at 400 K are listed in Table 1. It is evident that dc resistively increases with increase in Si concentration up to x = 0.15 and then decreases with increase in x. The activation energies (Eg) in ferrimagnetic and paramagnetic regions obtained from the plots of Log rdc and temperature are listed in Table1. The activation energy values in the paramagnetic regions are higher than that in ferrimagnetic region. This is in agreement with the theory developed by Irkhin and Turvo [12]. The values of Curie temperature (Tc) determined from the resistivity plots are listed in Table 1. It is evident that the Curie temperature decreases with increasing Si concentration DISCUSSION Decrease in lattice constant ‘a’ with increasing Si content from x = 0.1 to 0.4 show  that present samples obey Vegard’s law [4]. Usually in a solid solution of spinels within the miscibility range, a linear change in the lattice constants in observed [4]. The linear slow decrease in lattice constant in the present system can be explained on the basis of following facts.  (i)The larger Fe3+ ions (0.64 A0) are replaced by smaller Si4+ ions (0.42 A0) on A site so that ionic radius at A site decreases. (ii)The smaller Fe3+ ions (0.64 A0) are replaced by larger Co2+ (0.72 A0) on B site so that ionic radius of B site increases. (iii)The difference in ionic radii of Fe3+ and Si4+ (0.22 A0) on A Site is more than the difference in the ionic radii of Fe3+ and Co2+ (0.12 A0) on B site. Thus decrease in ionic radius of A site is greater than the increase in ionic radius of B site.       Due to complex structural composition of these ferrites, the conduction mechanism can be attributed to a combination of several processes. Fe2+ ions are formed due to partial reduction of small fraction of Fe3+ to Fe2+ ions due to volatilization of Zn at elevated firing temperature during sintering process [9]. In the present system, 2Fe3+ ions are replaced by Si4+ ions and Co2+ ions. Also CoO has an affinity for oxidation resulting in the formation of Co3+ ions. So increasing resistivity of the system with increase of Si content up x = 0.15 may be due to electron – hole compensation. The decrease in rdc with increase of Si content from x = 0.2 to 0.4 may be attributed to the presence of larger number of cobalt ions on B-site, which favor the hoping mechanism between Co2+ and Co3+ ions. Thus P-type, i.e. hole hoping between Co2+ and Co3+ in predominant conduction mechanism in the ferrites containing higher concentration of cobalt. These observations are consistent with the earlier reported results [10]. Variation of Logrdc with temperature (Fig.2) show decrease in resistivity with increasing temperature. This indicates semiconductor behavior having negative temperature coefficient of resistance. For higher concentrations of Si with x = 0.3 and 0.4, Log rdc is found to increase with temperature up to about 250 K. (Figure 2.) This anomalous variation of resistively at lower temperature for Si concentration x = 0.3 and 0.4 may be due to ionic drift current. The variation of rdc with temperature can be attributed to current due to electrons in conduction band, current due to electro hoping and ionic drift current [11]. The present results indicate that contribution from ionic drift current is more predominant in the anomalous region of rdc for samples with Si concentration x = 0.3 and 0.4. The ionic contribution becomes negligible at higher temperatures and rdc becomes entirely electronic. The activation energy (Eg) values are found to correspond to the dc resistivity values of the samples. The plots of Log rdc against reciprocal of temperature show abrupt change in the slope at the transition temperature. The change in the slope of the curves may be due to change in conduction mechanism [13] or it may be due to phase transition..                                                                             The decrease in Curie temperature Tc is because of net reduction of Fe3+ ions which reduces the number of active magnetic linkages                                                                         CONCLUSION Decrease in Lattice constant is due to replacement of larger ions by smaller ions. Decrease in X-ray density shows that decrease in mass overtakes decrease in volume of unit cell. Particle size is found to be consistent with the ceramically prepared ferrites. Increase in dc resistivity for lower concentration of Si is due to electron hole compensation while decrease in rdc at higher concentration of Si is due hole hoping between Co2+ and Co3+ ions. The anomalous variation of resistivity for higher concentration of Si is due to ionic drift current.                                                                           Acknowledgement      Author acknowledges the immense help received from the scholars whose articles are cited and included in references of 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. Conflict of Interest: Author declare no conflict of interest. Englishhttp://ijcrr.com/abstract.php?article_id=812http://ijcrr.com/article_html.php?did=8121.  M.G. Patil, V.C. Mahajan , A. K. Ghatege and S. A. Patil Indian J. of Pure and applied Phy.,34:166:1996 2.  V. C. Mahajan , Ph.D. Thesis, Shivaji University, Kolhapur (1995). 3.  S. S. Shinde , K. M. Jadhav , Mater Lett.,37: 63: 1998. 4.  C. G. Whinfrey , D. W. Eckart,  A. Taubar, J.Am. Chem. Soc.,82:2695:1960 5.  A. J. Miller, Appl. Phys., 30:248:1959. 6.  S.S. Shinde, K.M. Jadhav, G. K. Bichile, B. S. Trivedi and R. G. Kulkarni, Bull. Mat. Sci. 21:409:1998. 7.  M. J. Burger J. Crystal Structure Analysis, John Wiley, New York (1960). 8.  B. D. Cullity, Elements of X-ray diffraction, Addision Wesely, Reading, Mass, 1959. 9.  L. G. Van Uitert, Proc. IRE (USA), 1294 (1956) 1303. 10.A. B. Devale, D. K. Kulkarni, J. Pure and Appl. Phys. 16:697:1978. 11 R. H. J. Waldron, Appl. Phys. (USA), 43 (1972) 1186. 12.Yu.P. Irkin, E.A. Turov, Soviet Phys.JEPT, 33:673:1957. 13.K. R. Krishanamurthy, Thesis I.I.T., Madras (1975).

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524199EnglishN2017May23HealthcareElucidation of the role of the minC gene in filament formation by Listeria monocytogenes under stress conditions English0913Satyajit B. KaleEnglish Swapnil P. DoijadEnglish Krupali V. PoharkarEnglish Sandeep GargEnglish Ajay D. PathakEnglish Abhay V. RaoraneEnglish Deepak B. RawoolEnglish Nitin V. KurkureEnglish Sukhadeo B. BarbuddheEnglishAim: The study was conducted to understand structural changes in cell morphology of stress tolerant Listeria monocytogenes strains after exposure to the different food related stresses and to investigate the involvement of the minC gene in filament formation under stress as a putative mechanism. Methods: Morphological changes in L. monocytogenes were studied under the stresses of high salt concentration (12.5%), extreme pH (4.5 and 9.0) and low temperature (4°C). The structural changes were recorded employing light and electron microscopy. The expression of the minC gene under stress was studied by qPCR. Results: Long filament formations were observed under salt stress, while, no structural changes could be observed for isolates grown in extreme pH and low temperature stresses. Scanning electron microscopic studies showed 3-10 times elongation of cells under stress which got reverted to normal size after removal of stress. Interestingly, it was noted that with the increase in stress, rod shaped cells became elongated. Six to 11 fold expression of the minC gene was observed under high salt stress. Conclusion: The results suggested that the filament formation could be one of the mechanisms by bacteria to tolerate high salt stress. It also supported the hypothesis that the minC gene over-expression could be the factor behind filamentous morphology EnglishListeria mnocytogenes, Serogroups, Tolerance, Salt, minC, MorphologyINTRODUCTION Listeria monocytogenes is one of the 17 species of genus Listeria and is the cause of listeriosis in humans as well as in animals. It is an emerging foodborne pathogen with 20-30% case fatality rate, 50% neonatal death rate and 91% hospitalization rate (Sartor et al., 2015). Industrially processed and refrigerated foods revealed to be frequently linked to L. monocytogenes outbreaks than raw foods (Gianfranceschi et al., 2002). The pathogen has unique capabilities such as tolerance to high salt concentrations (> 10%) (Farber et al., 1992; Liu et al., 2005; Shabala et al., 2008), low temperature (down to 0°C) and diverse pH (pH 4.5 to 9.5) (Buchanan et al., 2004; Gandhi and Chikindas, 2007). Formation of filament (elongation of bacterial cell) is one of the stress responses under sublethal stresses (Giotis et al., 2007; Jones et al., 2013). This filament formation ability have been observed in L. monocytogenes under different stresses including high osmotic stress, low temperature, acidic and alkaline stresses (Bereksi et al., 2002; Giotis et al., 2007; Vail et al.,2012; Isom et al., 1995). The mechanism behind the filament formation under stresses has been studied in Gram negative bacteria, however, studies are largely lacking in Gram positive bacteria (Jones et al., 2013). It has been opined that when filamentous cells are exposed to more favorable growth conditions, there is rapid division of filaments into a number of individual cells (Giotis et al., 2007) which may pose major implications for the food industry. The potential numbers of viable bacteria maybe underestimated and may exceed tolerated levels in foods when filamentous cells that are subjected to sub lethal stress conditions (Jones et al., 2013). The formation of FtsZ septal ring, positioning of the ring, maturation of the ring and then cell division are key stages in bacterial cell division (Scheffers, 2008). This septal ring positioning which is controlled by two different systems namely Min system and nucleoid occlusion need to be on right place and on right time (Scheffers, 2008). The Min system prevents cell division at cell poles in which MinC and MinD proteins form an inhibitor which is topologically regulated by DivIVA (Rothfield et al., 2005). The MinC inhibits FtsZ ring formation at poles result of this is septation permitted at middle position of cell and other possible sites of cell division are blocked (Rothfield et al., 2005). Studies in Bacillus subtilis have shown that effect of MinC on FtsZ to be pH dependant (Scheffers. 2008). Therefore, it is hypothesized that under stressed environmental conditions the minC gene is over-expressed and inhibits the FtsZ ring formation at all possible sites which ultimately inhibits the septa formation in L. monocytogenes (Scheffers. 2008). Indian Listeria Culture Collection (ILCC) has a large collection of strains of Listeria isolated from various sources and diverse geographical areas of India. The collection has been screened previously for various stress responses and diversity. The objective of this study was to investigate the nature and dynamics of sublethal changes induced by stresses in high stress tolerant strains using scanning electron microscopy and to elucidate the role of the minC gene in L. monocytogenes under stressed conditions. MATERIALS AND METHODS Listeria monocytogenes strains Listeria monocytogenes strains (n=13) were selected from the Indian Listeria Culture Collection (ILCC) based on their tolerance to high salt, low temperature, low pH and high pH. The strains were characterized previously biochemically and for their serogroups (Doumith et al., 2004). All the strains were maintained at -80°C in brain heart infusion (BHI) broth (Himedia, India) with 15% sterile glycerol (v/v) (Himedia, India). Determination of morphological changes under stress The strains with the highest stress tolerance (high salt, low temperature, low pH and high pH) were selected to detemine the morphological changes under particular stressed environmental conditions. In brief, to determine the morphological changes by high salt stress, 16-18 h grown culture in BHI broth (2 ml) in 12.5% NaCl were centrifuged, washed twice with PBS to remove media particles, resuspended in 2 ml of PBS and a loopful of culture was taken on glass slide. The culture was stained by Gram staining and observed under compound microscope. Morphological changes were determined for strains in similar way after growth at different stresses of pH (4.5 and 9.0) and low temperature (4o C). Scanning electron microscopy (SEM) Strains exhibiting high tolerance for each of stresses (high salt concentration 12.5%, low temperature (4o C) and extreme pH of 4.5 and pH 9.5) were analysed for morphological changes using scanning electron microscopy (SEM). Bacterial strains were incubated in BHI broth (2 ml) under respective stress conditions. After 16-18 h, cell growth was harvested, washed twice with PBS, re-suspended in 2 ml of PBS and a loopful of culture was taken on cover slip and allowed to air dry. After air drying, the smear was fixed with 2.5% of glutaraldehyde for overnight and then dehydrated via successive passages of 10 minutes through 30%, 50%, 75%, 85%, 90%, 95% and 100% of ethanol. This preparation was then allowed to air dry and then sputter-coated with gold. The gold coated smears were examined by scanning electron microscope (EVO 18, Carl Zeiss, Germany). Determination of genetic basis of filament formation The minC gene in L. monocytogenes was detected by PCR in all the stress tolerant strains (n=13). The primers for amplification of the minC gene were designed using Primer 3 (ver. 4.0) software. Fifty microlitre reaction mixture consisting of 55 ng of bacterial genomic DNA, 15 pmol of each primer (F: 5’-AGA ACT AAC TCA ATT GCT TGC AG 3’ and R: 5’CAA ATC TGT TTC AGT GAC CTC TTT-3’), 25 µl of 2X PCR master-mix (Sigma, USA). The reaction was performed in an thermal cycler (Eppendorf, Germany) with initial denaturation at 95o C, 5.0 min; denaturation 95o C, 45 s; annealing 51o C, 30 s; and extension 72o C, 1 min. (35 cycles); final extension at 72o C, 10 min. PCR products were analyzed by electrophoresis in 1% agarose gels and visualized under G:Box gel documentation system (Syngene). Relative gene expression of the minC gene under high salt concentration The actual effect of salt stress on the minC gene expression was determined by qRT-PCR. The strains that formed filaments under salt stress were selected for the study. After growing the strains at mid-exponential phase under salt stress, RNA was isolated by using TRIzol reagent (Ambion, USA) following the manufacturers instruction. RNA yield was determined using the NanoDrop ND-1000 instrument (Thermo Scientific, USA) and RNA quality were checked by resolving it on 1.5% agarose gel. The cDNA synthesis was performed using SuperScript III First-Strand Synthesis  SuperMix for qRT-PCR (Invitrogen, USA). Two hundred ng of total RNA were converted into cDNA in 20 µL according to manufacturer’s protocol. The contamination of residual of DNA was checked in each RNA sample by a control reaction which includes a cDNA synthesis without reverse transcriptase enzyme (no RT control). The primers used in this reaction (Table 1) were designed by using Primer 3 software and optimized to achieve specific target gene amplification (product with a single melting peak). Quantitative-Real Time PCR mixture was prepared by using 10µL of 2x SYBR green master mix (Sigma, USA), 0.5 µL (10 nMol) of each forward and reverse primers 1µL of template cDNA and 8 µL of nuclease free water making volume to 20µL. As negative controls, water (no template) and the no RT control were applied. The reaction was performed in Light Cycler 96 (Roche, Switzerland). The reaction conditions were pre-incubation at 95o C for 10 min, then 40 cycles (95o C for 10s; 56o C for 20s and 72o C for 20s) followed by melting curve (65-97o C at 2.2o C/s and a continuous fluorescent measurement) and cooling. The expression of minC gene was normalized against expression of reference 23s rRNA gene (Romanova et al., 2006). The transcript levels of the minC gene were determined in strain exposed to high salt stress as well as optimal (no salt stress control) conditions. The fold induction of the minC transcript in response of high salt stress was calculated relative to optimal conditions transcript level. RESULTS Determination of morphological changes under stress Long filament formations were observed under salt stress (Fig. 1a and 1b), while, no structural changes could be observed for isolates grown under pH and temperature stresses. Therefore, effect on morphology under salt stress was further studied for longer time (till 72 h). With the increase in time, there was an increase in the length of filament (Fig. 1c). Compared to control, cells were present singularly (Fig. 1a) and few elongated cells were observed. Therefore, it appeared that under stress conditions there could be inhibition of cell division. To verify this phenomenon, the bacteria grown under salt stress (BHI with 12.5% of NaCl) were transferred to normal BHI broth and incubated for 2 h and observed for cell morphology. Interestingly, all the cells were observed to occur singularly (Fig.1d) as observed in control. Scanning electron microscopy Scanning electron microscopic studies showed 3-10 times elongation of cells under stress which got reverted to normal size after removal of stress. The length of the cell found to be increased (8.45 µm) (Fig. 2a) as compared to control (1.53µm) (Fig. 2b) under high salt stress. Under pH and low temperature stresses, there were no significant changes observed in morphology. The length of filament under high salt stress was observed to be increased (13.84 µm, 14.16 µm, 16.03 µm) (Fig. 2c and 2d) with the longer time of incubation up to 48 h to 72 h indicating a positive correlation (R²=0.9145) with filament formation with stress duration. Determination of genetic basis of filament formation under high salt stress The minC gene in L. monocytogenes was detected by PCR. All the 13 salt stress tolerant strains studied showed the presence of 475 bp band of the minC gene (Fig 3). The induction of the minC gene transcription level under high salt stress was examined by qPCR. All tested 13 strains showed the 6 to 11 fold expression of the minC gene under high salt stress (Fig. 4), suggesting the possible involvement of the minC gene in altered morphology with filamentous structure in L. monocytogenes under high salt stress. DISCUSSION The filament formation ability have been observed previously in L. monocytogenes under different stresses by different researchers including high osmotic stress, low temperature stress, acidic stress and alkaline stress (Bereksi et al., 2002; Giotis et al., 2007; Vail et al., 2012). It had been observed that as stress increased filamentation also increased (Giotis et al. 2007). These morphological changes under stress might infer that filament formation under stressed environmental conditions could be the one of the mechanisms of stress tolerance in L. monocytogenes. Changes in morphology of L. monocytogenes were observed after osmotic shifts. There was increase in length of the filament as with longer incubation (72 h). The bacteria regained normal size after removal of stress. This type of phenomenon was also observed earlier (Pratt et al., 2012). When it comes to the food processing and/or food preservation by using high salt bacteria which forms the filaments under stress could be the problem. Since after exposure to the less stressed conditions filamentous cells immediately divides in individual cells, ultimately there is an increase in the number of bacteria in the food (Bereksi et al., 2002; Jones et al., 2002). The formation of filamentous cells leads to forming a single colony on solid media. This may results in a false estimation of bacteria in particular food product by cultural methods and false readings in predictive models for growth kinetics of bacteria based on CFU methods (Giotis et al., 2007) significantly increasing the risk to consumers. Though filament formation under stress has been observed in L. monocytogenes, however, studies are largely lacking (Jones et al., 2013). L. monocytogenes is normally exposed to various stresses during food processing and disinfection procedures which could influence its response and ability to persist in these environments, and thus contribute to defining conditions for better control in food processing plants (Magalhaes et al., 2016). There was over-expression of the minC gene observed in strain with the filamentous morphology under high salt stress. It supported the hypothesis that the minC gene overexpression could be the factor behind filamentous morphology of L. monocytogenes under high salt stress and forming a filament could be the one of the mechanisms by bacteria to tolerate high salt stress. As it was preliminary study performed by taking single test strain, it would be more evident after testing more number of strains with filament forming ability for same hypothesis. CONCLUSION It is concluded that the filament formation under stressed environmental conditions could be the one of the mechanisms of stress tolerance in L. monocytogenes as evidenced by morphological analysis of strains under stress. This study is a significant step towards dissecting the stress response of L. monocytogenes to the molecular events. Further investigations are required at molecular level to understand mechanism of filament formation, this will help towards better understanding of growth and survival properties of these filament and improvements in safety and risk assessment of food products. 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. The research work is supported by grants from the Department of Biotechnology, Government of India (BT/01/CEIB/11/VI/13) to SBB and NVK. Source of Funding The Department of Biotechnology, Government of India to SBB and NVK. Conflict of interests None to declare.   Englishhttp://ijcrr.com/abstract.php?article_id=813http://ijcrr.com/article_html.php?did=8131. Bereksi N, Gavini F, Bénézech T, Faille C.. Growth, morphology and surface properties of Listeria monocytogenes Scott A and LO28 under saline and acid environments. J Appl Microbiol 2002; 92: 556–565. 2. Buchanan R, Lindqvist R, Ross T, Smith M, Todd E, Whiting R.. Risk assessment of Listeria monocytogenes in ready-to-eat foods. Microbiological Risk Assessment Series, 4. Food and Agriculture Organization of the United Nations, 2004. 3. Doumith M, Buchrieser C, Glaser P, Jacquet C, Martin P. Differentiation of the major Listeria monocytogenes serovars by multiplex PCR. J Clin Microbiol 2004; 42: 3819-3822. 4. Farber JM, Coates F, Daley E. Minimum water activity requirements of the growth of Listeria monocytogenes. Lett Appl Microbiol 1992;15:103-105. 5. Gandhi M & Chikindas M L.. Listeria: a foodborne pathogen that knows how to survive. Int J Food Microbiol 2007;113:1–15. 6. Gianfranceschi M, Gattuso A, Tartaro S, Aureli P. Incidence of Listeria monocytogenes in food and environmental samples in Italy between 1990 and 1999: Serotype distribution in food, environmental and clinical samples. Eur J Epidemiol 2002;18: 1001–1006. 7. Giotis E S, Blair IS, McDowell DA. Morphological changes in Listeria monocytogenes subjected to sublethal alkaline stress. Int J food Microbol 2007;120:250–258. 8. Isom L L, Khambatta ZS, Molus J L, Akers DF, Martin S E. Filament formation in Listeria monocytogenes, J Food Prot 1995; 58:1031–1033. 9. Jones T, Gill C O, McMullen L, The behavior of log phase Escherichia coli at temperatures below the minimum for sustained growth. Food Microbiology 2002; 19:83–90. 10. Jones TH, Vail KM, McMullen LM. Filament formation by foodborne bacteria under sublethal stress. Inter J Food Microbiol 2013;165: 97–110. 11. Liu D, Lawrence ML, Ainsworth A J, Austin FW. Comparative assessment of acid, alkali and salt tolerance in Listeria monocytogenes virulent and avirulent strains. FEMS Microbiol Lett 2005;243: 373-378. 12. Magalhaes R, Ferreira V, Brandão T R, Palencia RC, Almeida G, Teixeira P. Persistent and non-persistent strains of Listeria monocytogenes: A focus on growth kinetics under different temperature, salt, and pH conditions and their sensitivity to sanitizers, Food Microbiol 2016;57:103-108. 13. Pratt AL, Chen B, Czuprynski CJ, Wong ACL, Kaspar CW. Characterization of osmotically induced filaments of Salmonella enterica. App Enviro Microbiol 2012;78:6704–6713. 14. Rothfield L, Taghbalout A and Shih, Y.L., Spatial control of bacterial division-site placement. Nature Rev Microbiol 2005; 3:959–968. 15. Sartor C, Grégoire E, Albanèse J, Fournier PE. Invasive Listeria monocytogenes infection after liver transplantation: a lifethreatening condition, Lancet. 2015;6736: 61831-61836. 16. Scheffers D -J. The effect of MinC on FtsZ polymerization is pH dependent and can be counteracted by ZapA. FEBS Letters 2008;582: 2601–2608. 17. Shabala L, Lee SH, Cannesson P, Ross T.. Acid and NaCl limits to growth of Listeria monocytogenes and influence of sequence of inimical acid and NaCl levels on inactivation kinetics, J Food Prot 2008;71:1169-1177. 18. Vail KM, McMullen LM, Jones TH. Growth and filamentation of cold-adapted, log phase Listeria monocytogenes exposed to salt, acid, or alkali stress at 3°C. J Food Prot 2012; 75:2142– 2150.  

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524199EnglishN2017May15HealthcareManagement of Subtrochanteric Fractures with Long PFN: Union Rates and Functional Results English1419Shakeel AhmadEnglish Rajinder SinghEnglish Abdul GhaniEnglish Khalid MuzaffarEnglish Omeshwar SinghEnglishIntroduction: The treatment of subtrochanteric fractures has evolved over a period of time, from conservative to extramedullary fixation to intramedullary fixation. The management of these fractures poses a significant challenge, the criteria for functional recovery becoming more and more exacting. Aim of study: To assess union rates and functional status after long PFN in subtrochanteric fractures. Material & Methods: A prospective study on adult patients with a follow up of 6 months was conducted at Orthopaedic department of Govt. Medical College Hospital, Jammu between June 2014 and May 2016. 40 patients above the age of 18 years with subtrochanteric fractures were included in the study. Patients were assessed clinically and radiologically on the 2nd postoperative day, at 4 weeks, 12 weeks and then between 6 months to 1 year depending upon the fracture union. At each follow up, functional evaluation of the patient was done. Results: Majority of fractures(72.5%) united within 14 weeks.Full weight bearing was allowed by 12th week in majority (57.5%) of cases.Salwati & Wilson hip function score improved from 16 at discharge to 36 at 6 month follow up. As per Kyle’s criteria, results achieved were Excellent/Good in 95% of cases. Conclusion: Proximal femoral nail is an efficient device for the treatment of subtrochanteric fractures with high rate of bony union. The great majority of patients were provided with stable fixation, early mobilization, early rehabilitation and return to pre-fracture status. EnglishSubtrochanteric fractures, Long PFN, Union rates, Kyle’s criteriaINTRODUCTION The treatment of subtrochanteric fractures has evolved over a period of time, from conservative to extramedullary fixation to intramedullary fixation. The high incidence of delayed union, malunion and nonunion of fractures in this region has left conservative treatment; as advocated by DeLee et al1 abolished in modern trauma care. This evolution in treatment modalities is due to unsatisfactory results achieved in these fracturesdue to medial comminution, high tensile forces active in this region2 and various muscular deforming forces acting on the fracture fragments3thus leading to high incidence of malunion and nonunion in these fractures and mechanical failure of the implant4,5. The appropriate implant for the internal fixation of subtrochanteric fractures remains debatable; and a multitude of different intra- and extramedullary devices for their surgical fixation have been advocated6- 10. The advantage of closed reduction over open reduction cannot be over emphasized. Open reduction increases the risk of infection, soft-tissue devitalisation, and non-union and thus, poor functional results. Up to one half of the patients with subtrochanteric fractures may not regain their pre-fracture walking capacity, and independent living may no longer be possible11. The management of these fractures poses a significant challenge, the criteria for functional recovery becoming more and more exacting. Subtrochanteric fractures are predisposed to complications from loss of position including coxa vara, rotational deformity or shortening, non-union due to implant breakage or failure of bone substance and migration of fixation components12. We now report the results of clinical and radiographic follow-up of a series of subtrochanteric fractures treated with long proximal femoral nail (PFN – AO type design). AIM OF STUDY To study union rates after long PFN in subtrochanteric fractures To assess functional status using Salwati & Wilson hip function scoring system after PFN in subtrochanteric fractures MATERIAL AND METHODS A prospective study on adult patients of both sexes with a follow up of 6 months was conducted at Orthopaedic department of Govt. Medical College Hospital, Jammu between June 2014 and May 2016.40 patients above the age of 18 years with subtrochanteric fractures were included in the study. After clinical assessment and haemodynamic stabilization, patients were subjected for radiographs of Pelvis with both hips-AP view and full length thigh-AP and lateral views. Patients selected for study include: Age - above 18 years Sex - both males and females. Patients with Seinsheimer types I –IV subtrochanteric fractures Patients excluded from study include: Polytrauma Old complicated fractures. Pathological fractures. Seinsheimer type V subtrochanteric fractures. Subtrochanteric fractures with associated neck/intertrochanteric/shaft fractures. Patients with any contraindication for anaesthesia or surgery All the patients were operated on a fracture table in supine position under image intensifier control using standard techniques. Patients were assessed clinically and radiologically on the 2nd postoperative day, at 4 weeks, 12 weeks and then between 6 months to 1 year depending upon the fracture union.At each follow up, functional evaluation of the patient was done to note the range of movements at the hip and knee, ability to walk, any pain, limp, residual shortening, deformities, wound condition and any residual complaint. Healing was judged by both clinical (pain & motion at fracture site) and radiological (bridging callus filling the fracture site or trabeculations across the fracture site) criteria and functional outcome was reviewed according to the Salwati and Wilson hip function scoring system. The final assessment was carried out at 24 weeks on the basis of radiological union and Salwati and Wilson hip scoring system. Radiological assessment:- All radiologically ununited fractures were graded as poor results. Salwati and Wilson hip scoring system:- The evaluation of results was based on following 4 parameters as Pain, Walking, Muscle power& motion, and Function. The results were classified as Excellent when the sum of scores was 32 or more, Good when it is 24 to 31, Fair when it is 16 to 23 and Poor when the score is 15 or less. Pain 0 –Constant and unreliable, frequent strong analgesia 2 – Constant but bearable, occasional strong analgesia. 4 – Nil or little at rest, pain on activity. 6 – Little pain at rest, pain on activity. 8 – Occasional slight pain. 10 – No pain. Walking 0 – Bed ridden 2 – Wheel chair 4 – Walking frame 6 – One stick, distance limited up to 400 yards 8 – One stick, long distance 10 – Unaided and unrestricted Muscle power and Motion 0 – Ankylosing and deformity 2 – Ankylosing with good functional position 4 – Poor muscle power, flexion less than 60 degrees, abduction less than 10 degrees 6 – Fair muscle power, flexion 60 – 90 degrees, abduction 10 – 20 degrees. 8 – Good muscle power, flexion more than 90 degrees, abduction more than 20 degrees. 10 – Normal muscle power, full range motion Function 0 – Bed ridden 2 – House bound 4 – Limited house work 6 – Most house work 8 – Very little restriction 10– Normal activity. RESULTS Total no. of patients in our study were 40; of which 25 were males and 15 females. Most of the patients belonged to age group of 41 – 50 years. The fractures were classified according to Seinsheimer classification13 (type II- 22 cases, type III – 14 cases, type IV – 4). In most of the cases(n = 23), the mechanism of trauma was a Road traffic accident, while as, in rest of cases(n = 17), Fall from height was responsible for trauma. 50% cases had associated injuries like fracture distal end radius, fracture calcaneum etc. Other characteristics noted are as mentioned below: From the chart, it is clear that majority of cases were managed by closed reduction (87%). 8 % cases required mini-open reduction. In 92% of cases, reduction achieved was anatomical to acceptable. Whereas, in 8% of cases, reduction was not acceptable NEED FOR BONE GRAFTING None of the cases required bone grafting in our series. All fractures united spontaneously. Table 1 shows that majority of fractures united within 14 weeks(72.5%). Table 2 shows that in 75% of cases, we started toe touch weight bearing within 4 days. Table 3 shows that partial weight bearing was started at 4th week in majority of cases (40%). Table 4 shows that we had allowed full weight bearing by 12th week in majority (57.5%) of cases.  Table 5 shows that excellent/good range of movement was achieved in majority (80%) of cases. Table 6 shows that good range of movement was achieved in 85% of cases. Table 7 shows that 80% of cases reported no pain at 24 week follow up. Table 8 shows that 95% of patients attained good/normal muscle power by 6 months. Table 9 shows that 90% of patients had attained normal walking ability. Table 10 shows that we achieved Excellent/Good results in 95% of cases. Pre-operative and Post-operative radiograph of40 year old patient. Patient is shown as sitting cross legged at 6 month follow up. DISCUSSION Subtrochanteric fractures of the femur have peculiar anatomic and mechanical characteristics which poses problems in their management. Extramedullary fixation with plating has the potential disadvantages of extensive surgical exposure, severe soft tissue damage and blood loss, thus leading to problems of fracture union and implant failure. In addition, the eccentric plating is prone to fatigue breakage due to their mechanical load-sharing effect14. Allowing a minimally open approach, intramedullary nailing is closely linked to “biological internal fixation”, in addition to its mechanical benefits over plate fixation15. The result of the reduction was considered acceptable in 55% of the patients and anatomical in 37% of patients. Poor reduction was noted in 8% of patients and it was associated with poor outcome. In SCHIPPER et alstudy16, reduction was good to acceptable in 96.2% of their patients and poor reduction was seen only in 2.9% of their patients. Most of the cases in our series were managed by close reduction. However, in 8% of cases, mini-open reduction was required. Here, a small incision was made over the fracture site without extensive soft tissue dissection and using bone levers or bone clamps, fragments were held in reduced position till nailing was done. This is comparable with the studies of Boldin et al17(90.90% Close Reduction) and other studies done elsewhere. Varus malreduction was the most common complication noticed in our series. This can be attributed to initial malreduction in which proximal fragment remained abducted. It is important that fracture should be reduced first, even if open reduction is required, before intramedullary nailing. In this study, clinical union was achieved within 14 weeks in 72.5% of cases which is comparable to Smith et al series, who reported clinical union in 10.5 weeks and to Domingo et al series who reported union in 12 weeks. In our study, one patient had iatrogenic femoral shaft fracture which took24 weeks to unite. In other series, the rate of fixation failure, femoral shaft fracture, and re-operation rate is high18 (12 %).                The mobilization strategy followed; touch down weight bearing was allowed within 4 days in 75 % of cases in whom there was little or no cortical comminution pre-operatively and in whom solid fixation was achieved, whereas it was allowed on 5th or 6th day in 20% of cases and after 6th day in 5% of cases where the fixation was not solid or there was gross comminution or due to iatrogenic fracture. This is consistent with the study of Smith et al, who allowed touch down weight bearing on 2nd postoperative day in 71.5% of cases and delayed in 28% of cases. In our series, average time to full weight bearing was 14 weeks in 87.5% of cases which is late as compared to the series by Smith et al who started full weight bearing by 6.7 weeks.Walking and squatting ability was completely restored in each case at follow-up examination 6 months postoperatively.   Englishhttp://ijcrr.com/abstract.php?article_id=814http://ijcrr.com/article_html.php?did=814 DeLee JC, Clanton TO, Rockwood CA Jr. Closed treatment of subtrochanteric fractures of the femur in a modified cast-brace. J Bone Joint Surg Am 1981;63:773-9. Koch JC. The laws of Bone architecture. Am j anat 1917; 2:177. Campbell’s operative orthopaedics. 12th edition, Vol 3, 2751 – 2752, 2013. Haidukewych GJ, Berry DJ. Non-union of fractures of the Subtrochanteric region of the femur. Clin Orthop Relat Res 2004;419:185-8 Broos PL, Reynders P. The use of the unreamed AO femoral intramedullary nail with spiral blade in nonpathologic fractures of the femur: experiences with eighty consecutive cases. J Orthop Trauma 2002;16:150-4. Blatter G, Janssen M. Treatment of subtrochanteric fractures of the femur: reduction on the traction table and fixation with dynamic condylar screw. Arch Orthop Trauma Surg 1994;113:138-41. Hotz TK, Zelweger R, Kach KP. Minimal invasive treatment of proximal femur fractures with the long gamma nail: indication, technique, results. J Trauma 1999;47:942-5 Tornetta P III. Subtrochanteric femur fracture. J Orthop Trauma 2002;16:280-3 Siebenrock KA, Muller U, Ganz R. Indirect reduction with a condylar blade plate for osteosynthesis of subtrochanteric fractures. Injury 1998;29Suppl 3:C7-C15. Ruff ME, Lubbers LM. Treatment of subtrochanteric fractures with a sliding screw-plate device. J Trauma 1986;26:75-80. Koval KJ, Skovron ML, Aharonoff GB, Zuckerman JD. Ambulatory ability after hip fracture. A prospective study on geriatric patients. Clin OrthopRelat Res.1995;310:150-9 Roberto U, Velasco, MD; and Thomas H. Comfort, MD. Analysis of treatment problems in sub-trochanteric fracturesof the femur: The Journal of Trauma, Vol. 18, No. 7, 513 – 523, 1978. Seinsheimer, F., III: Subtrochanteric fractures of the femur. J.Bone Jt. Surg., 60-A: 300-306, 1978. Lunsjo K, Ceder L, Thorngren KG, Skytting B, Tidermark J, Burntson PO. Extramedullary fixation of 569 unstable intertrochanteric fractures. Acta Orthop Scand 2001;72:133-40. Brien WW, Weiss DA, Becker V Jr, Lehman T. Subtrochanteric femur fractures: a comparison of the Zickel nail, 95-degree blade plate, and interlocking nail. J Orthop Trauma 1991;5:458-64 Schipper I B et al Treatment of Unstable trochanteric fractures :JBJS 2004; 86 B : 86 – 94 Christian Boldin, Franz J Seibert, Florian Fankhauser: "The proximal femoral nail (PFN)—-a minimal invasive treatment of unstable proximal femoral fractures. Acta Orthop Scand 2003; 74(1): 53 - 58. Menezes DF, Gamulin A, Noesberger B. Is the proximal femoral nail a suitable implant for treatment of all trochanteric fractures? Clin Orthop Relat Res 2005; 439:221–7. Charnley J. The long-term results of low-friction arthroplasty of the hip performed as a primary intervention. J Bone Joint Surg Br 1972;54:61-76 Strömberg L, Lindgren U, Nordin C, Ohlen G, Svensson O. The appearance and disappearance of cognitive impairment in elderly patientsduring treatment for hip fracture. Scand J Caring Sci 1997;11:167-75. Huusko TM, Karppi P, Avikainen V, Kautiainen H, Sulkava R. Randomised, clinically controlled trial of intensive geriatric rehabilitation in patients with hip fracture: subgroup analysis of patients with dementia. Bmj 2000;321:1107-11. Svensson O, Strömberg L, Ohlen G, Lindgren U. Prediction of the outcome after hip fracture in elderly patients. J Bone Joint Surg Br 1996;78:115-8.

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524199EnglishN2017May15HealthcareFormation and Reversion of VBNC Cells of Salmonella Typhimurium Preincubated in Different Substrates English2025Nadezhda SkorlupkinaEnglish L. BlinkovaEnglish Yu. PakhomovEnglish A. PiyadinaEnglish D. ChistaykovaEnglishObjectives: Salmonella is one of the main dangerous opportunistic microorganisms, and it can persist in viable but nonculturable (VBNC) state in different foodstuffs, water, human or animal organisms etc. Thus it is important to know more about this form of bacterial existence. Methodology: In this study Salmonella Typhimurium 79 strain was used, in which RP4 plasmid was introduced. Prior to entering nonculturability inducing conditions cells of the strain were preincubated in one of the substrates that are typical sources of Salmonella contamination. Several additives and conditions were evaluated in order to revert nonculturable Salmonella into vegetative state. Results: Influence of preincubation in different substrates on speed and totality of entry into nonculturable state was determined. It was shown that preincubation in soil had the greatest impact on the Salmonella population. Control population, preincubated in nutrient broth, was the slowest to enter nonculturable state. Tested reversion factors had effects at different concentrations. The most efficient, though the slowest, way to revert Salmonella was to incubate VBNC cells in 0.9% NaCl. Conclusion: Obtained data reveal some reasons for strain differences in speed of formation of VBNC cells. Reversion was achieved using several supplements and conditions. EnglishNonculturable, Factors, Opportunistic microorganismsIntroduction The viable but nonculturable (VBNC) state is defined as a state in which microorganisms are metabolically active but lack the ability to reproduce on routine culture media after stress. (Oliver, 2005) If bacteria in VBNC state can revert to physiologic activity, they will be considered alive and will have metabolic activity. (Oliver, 2000; Pakhomov Yu, et al., 2016) Many bacteria, including human pathogens, can enter into the VBNC state in presence of unfavorable environmental conditions. (Roszak D. B. et al, 1984; Oliver J.D. 2010) Transition of pathogenic microorganisms into a resting state is one of the basic mechanisms permitting the preservation of an infectious causative agent in the environment during interepidemic seasons or maintaining endemic area. (Bukharin O.V. et al, 2005) Cells enter VBNC state as a response to some form of natural stress, such as starvation, incubation outside the temperature range of growth, elevated osmotic concentrations (seawater), reactive oxygen concentration, exposure to white or UV light (Oliver, 2005; Shenghua Zhang, 2015). Every factor shifts bacteria into VBNC state with different speed, intensity, and launches various genetic mechanisms. (Bukharin O.V. et al, 2005) It is important to learn how bacteria enter VBNC state in different nature objects. Bacteria in VBNC state don’t reproduce on standard culture media (Oliver, 2010). It is the main problem in research – contamination of different objects, e.g. foodstuffs, drinking water, human clinical specimens, etc. with nonculturable bacteria. Methodology Strains and culture conditions For experiments Salmonella enterica serotype Typhimurium 79 was used with a plasmid RP4 that has resistance genes for ampicillin, tetracycline and kanamycin. Microbe was precultured in 100 ml of nutrient broth with 100 µg/ml of ampicillin and 50 µg/ml of kanamycin for 24 hours twice and then a population was produced on the same medium for 20 hours. Samples of possible Salmonella sources were inoculated with this population in an amount of 1% of the medium. These samples were: wet soil (substrain S), tap water (W), fresh vegetable salad (cucumbers, tomatoes, black olives, bell pepper and sunflower oil; substrain V) and minced chicken (P). They were not sterilized in any way to keep them natural as much as possible. Fresh nutrient broth without antibiotics (C) was chosen as the control substrate. All the samples were incubated for 7 days. Incubation conditions were mimicked those of natural sources of Salmonella. Water and soil samples were stored at room temperature, foods – at 0°C in the refrigerator and control media – in thermostat at 37 °C. After incubation bacteria were isolated from samples on nutrient agar with the same amounts of antibiotics. Salmonella were transferred into 200 ml of commercial artificial sea water (ASW). This medium was purchased in a local aquarium store, prepared according to manufacturer’s instructions and sterilized for 15 minutes at 121°C. ASW’s flasks with Salmonella were incubated in stationary conditions at room temperature in the darkness. Samples were taken periodically and plated on nutrient agar without antibiotics and on nutrient agar diluted fivefold. Reversion of nonculturable cells For reversion Salmonella in VBNC state for 8 month (C) was used. Stimulating supplements were inulin (powder, "Ryazanskie Prostory", Russia), Helianthus tuberosus powder with 70% inulin content (powder, “Eco-Pro”, Russia), vitamin C (ascorbic acid) (injection 50mg/ml, “Escom”, Russia), vitamin PP (nicotinic acid) (injection 10mg/ml, “RENEWAL”, Russia) and blood-substitute Aminopeptid (liquid, “Samson-Med”, Russia). Every supplement was added to nutrient broth at different concentrations: for inulin or Helianthus tuberosus powder it was 0.1%, 1% or 10%; for vitamin C or vitamin PP it was 0.01%, 0.05% or 0.1%; for blood-substitute it was 5%, 10% or 20%. Control was inoculated in nutrient broth without any supplements. The samples with and without supplement were inoculated with 1% Salmonella culture from one. Then they were incubated for 48 hours in thermostat at 37 °C. Every 24 hours, tests were taken and bacterial VBNC state was analyzed. Also in order to revert cells of all substrains suspensions were serially diluted in 0.9% NaCl up to 10-8. Diluted populations were incubated for 96 hours. Samples were taken periodically for CFU/ml assessment. Viability and culture ability assays For every sample (the first or the second part) viability and culturability were monitored. Total cell counts (TCC) were performed in Goryaev-Thoma counting chamber. Portions of viable cells were assessed under a luminescence microscope (OPTON, 8×40-time magnification) after staining with Live\Dead® double staining kit (Baclight™). Bacteria were plated on nutrient agar, plates were incubated in thermostat at 37 °C for 24 hours and CFU/ml values were defined. The number of VBNC cells was calculated as difference between amounts of viable cells and CFU/ml. Statistical Methods The data was analyzed by parametric statistical methods. Results Initial cell concentrations for all populations were about 4×107 cells/ml. During first week CFU/ml value lowered by one order of magnitude for all samples except C. At this point the lowest index was observed for the sample W (Table 1). After 3 weeks of incubation the lowest CFU/ml value was noted for the sample P – 3 orders of magnitude lower than initial value. In other populations it dropped by 2 orders of magnitude. Sample showed C the highest CFU/ml value for the first 3 months of incubation After 9 weeks population S exhibited only singular colonies which means that it is almost totally nonculturable. After 3 months this substrain started exiting nonculturable state. Viability was tested using a luminescence microscope. It was shown that cells retained their viability for at least 8 months. Total cell counts remained relatively constant throughout the entire observation period (Table 2). However it was noted that in all populations cells became smaller and fluorescence grew dimmer. (Fig. 1) Since it was shown that some microorganisms are able to resuscitate from nonculturable state when stressful factor is withdrawn (Su C–P. et al. 2013) we tried to use normal saline (isotonic protein-free poor medium) to resuscitate our bacteria. The following dynamics were noted for resuscitation of Salmonella in our experiment: after 6 hours there was no noticeable difference when compared to cells in artificial seawater. After 24 hours for samples C and S 2 orders of magnitude increase in culturability was noted (from 3.62±0.32x105 to 5.13±0.49x107 and from 3.72±0.34x103 to 3.12±0.29x105 respectively. For samples V and P difference was 1 order of magnitude from 1.6±0.15x106 to 1.7±0.15x107 and from 1.3±0.12x105 to 5.9±0.52x106 respectively. There was no noticeable increase for the sample W at this point (Table 3). After 48 hours CFU/ml value increased by 2 more orders of magnitude for C and V and by 3 more orders of magnitude for S, P and W. After 96 hours resuscitation process was completed. Increased CFU/ml values after resuscitation may be explained by some amount of cellular growth and division of resuscitated cells even in poor environment of normal saline. After incubation for 96 hours in normal saline cells regained bright fluorescence that was noted in the beginning of the experiment which means that their metabolism probably returned to normal state and DNA decondenced. It was also noted that cells of S variant did not divide as actively as the rest of studied variants. It should be noted that we incubated Salmonella as serial decimal dilutions up to 10-8 and noted active growth after 24 – 96 hours in samples from tubes that showed singular or no colonies when plated immediately after dilution. Therefore such increase in numbers of CFU/ml cannot be attributed to division of remaining culturable cells. For reversion Salmonella of population C was used which had been in VBNC state for 8 months. The amount of live bacteria was initially 99%, the amount of bacteria in VBNC state was 97±2%. The content of inulin at 1% in nutrient broth Salmonella reversion appeared on the first day – 37% (CFU/ml - (1,7±0,21)x108, TCC/ml - (2,68±0,19)x108) cells in VBNC state, control – 90% (CFU/ml – (1,5±0,16)x107, TCC/ml - (1,48±0,11)x108 ). (Fig. 2, a) At 48 hours the number of bacteria in VBNC state was decreased to 13% (CFU/ml – (1,7±0,26)x109, TCC -(1,96±0,13)x109), control – 80% (CFU/ml – (1,6±0,68)x108, TCC/ml - (8,6±0,57)x108). At other concentrations significant differences were not observed. The content of 1% Helianthus tuberosus powder in nutrient broth Salmonella reversion was registered only on the second day (48 hours) – 7% (CFU/ml - (4,1±0,87)x109, TCC/ml - (4,4±0,31)x109) cells in VBNC state, control – 81% (CFU/ml - (3,4±0,43)x108, TCC/ml - (1,8±0,13)x109 ). (Fig. 2, b) At other concentrations significant differences were not observed. The content of 0.01% vitamin PP in nutrient broth Salmonella reversion was happened on the second day (48 hours) – 13% (CFU/ml - (1,01±0,15)x109, TCC/ml - (1,16±0,1)x109) cells in VBNC state, control – 82% (CFU/ml - (2,01±0,29)x108, TCC/ml - (1,1±0,1)x109). (Fig. 2, c) At other concentrations significant differences were not observed. At all concentrations vitamin C or blood-substitute Aminopeptid positive effect was not found for Salmonella reversion of the VBNC state in the vegetative cells. (Fig. 2, d) Discussion Obtained data suggest that preincubation of S. enterica Typhimurium 79 in different substrates before entry into stressful environment influences its speed of entry into nonculturable state. Preincubation in soil had the biggest influence on the microorganism. However, with time bacteria adapted to the stressful environment of the artificial seawater and numbers of CFU/ml stabilized. Sample S exhibited the least CFU/ml value in the second half of the incubation period. At the same time sample C incubated in rich optimal environment of nutrient broth had the highest culturability for the first three months. These differences caused by environmental conditioning in our opinion shed some light on question why there is a strain difference in speed and sometimes even possibility of entry into nonculturable state (Chaveerach P., 2003, Masuda Y., 2004). We believe that after three months of incubation in artificial seawater there was some adaptation to the osmotic stress and numbers of CFU/ml somewhat stabilized and never dropped below 4.5?104 except for the sample S. Incubation in stressful conditions without aeration did not result in notable cell death. This is an expected result since oxidative stress is one of the major factors that promote entry into nonculturable state (Kong I.–S. et al., 2004). Dwarfing of bacterial cells is one of the properties of nonculturable cells (Oliver J.D. 2010) and dimming of fluorescence may be due to slowed metabolism and condensing of cell DNA which makes it less available for DNA binding dyes. In our experiments we observed some spontaneous resuscitation of bacterial cells. This phenomenon is most clearly visible for the sample S. This is probably due to presence of some stimulating agents from a minute portion of lyzed cells or cell adaptation to stressful environment. After removing osmotic stress, we observed complete resuscitation in all sample populations. Increased CFU/ml values after resuscitation (compared to total viable counts in flasks with artificial seawater) may be explained by some amount of cellular growth and division of resuscitated cells even in poor environment of normal saline. After incubation for 96 hours in normal saline cells regained fluorescence as bright as that was noted in the beginning of the experiment which means that their metabolism probably returned to normal state and DNA decondenced. It was also noted that cells of S variant did not divide as actively as the rest of studied variants and the control population was the most active. It should be mentioned that we incubated Salmonella in normal saline as serial decimal dilutions of artificial seawater up to 10-8 and noted high CFU counts after 24 – 96 hours in aliquots from tubes that showed singular or no colonies when plated immediately after dilution. Therefore, such increase in numbers of CFU/ml cannot be attributed to division of remaining culturable cells. Helianthus tuberosus powder or inulin at 1%, and 0.01% nicotinic acid in nutrient broth significantly accelerated the reversion of Salmonella from VBNC into active state. Probably small concentrations are insufficient to produce a positive effect, but large doses may manifest the inhibitory effect. During incubation with different concentrations of blood-substitute Aminopeptid or ascorbic acid we did not observe positive effect of the reversion of VBNC into active state. Perhaps Salmonella in VBNC state is not sensitive to these agents, or the factors’ concentrations were not optimal. The next problem is to optimize the amount of inulin or nicotinic acid by which better and faster reversion is promoted. Helianthus tuberosus powder is unsuitable for experimental work. Conclusion Thus it was established that preincubation in different substrates influenced speed of formation of nonculturable cells which may be one of the reasons for strain differences in this process. Also reversion of nonculturable cells was achieved using different supplements and conditions. 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 None declared Conflict of interest None declared Fig. 1. Salmonella enterica serotype Typhimurium 79 cells. Left column – cells at the first day of incubation, Right column – cells after 8 months.   Englishhttp://ijcrr.com/abstract.php?article_id=815http://ijcrr.com/article_html.php?did=815 Bukharin O.V., Gintsburg A.L., Romanova Y.M., El-Registan G.L. Mechanisms of survival bacteria. Moscow: Medicina, 2005; 367p (in russian). Chaveerach P., A. A. H. M. ter Huurne, L. J. A. Lipman, and F. van Knapen Survival and resuscitation of ten strains of Campylobacter jejuni and Campylobacter coli under acid Conditions Appl and Environ Microbiol, 2003; Vol 69, ? 1: 711–714 Kong I.–S., A. Hülsmann, T.C. Bates, J.D. Oliver. The role of reactive oxygen species in the viable but nonculturable state in Vibrio vulnificus. FEMS Microbiol. Ecol. 2004; Vol. 50: 133–142. Masuda Y. Resuscitation of Tenacibaculum sp., the causative bacterium of spotting disease of sea urchin Strongylocentroutus intermedius from viable but non–culturable state. / Y. Masuda, K. Tajima, Y. Ezura Y. // Fisheries Science. 2004; Vol. 70(2): 277–284. Oliver, J.D. The public health significance of viable but nonculturable bacteria, In R.R. Colwell and D.J. Grimes (eds.), Nonculturable Microorganisms in the Environment. American Society for Microbiology Press, Washington, D.C.;2000: 277-299. Oliver, J. D. The viable but nonculturable state in bacteria. J. Microbiol.2005;43: 93–100 Oliver J.D. Recent findings on the viable but nonculturable state in pathogenic bacteria. FEMS Microbial Rev. 2010. Vol. 34(4), P. 415–425. Pakhomov Yu.D., L.P. Blinkova, O.V. Dmitrieva, O.S. Berdyugina, N.N. Skorlupkina Resuscitating factors for nonculturable cells. Psysical chemistry for the chemical and biochemical sciences, April 2016; 335-342 Roszak, D. B., Grimes, D. J., & Colwell, R. R. Viable but nonrecoverable stage of Salmonella enteritidis in aquatic systems. Canadian journal of microbiology, 1984; 30(3): 334-338. Shenghua Zhang, Chengsong Ye, Huirong Lin, Lu Lv, and Xin Yu*. UV Disinfection Induces a VBNC State in Escherichia coli and Pseudomonas aeruginosa Environ. Sci. Technol., 2015; 49(3): 1721-1728

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524199EnglishN2017May15TechnologyAwareness and Need of Ethics and Values in Education for Students: A Study among College Teachers in Pollachi Region English2631S. ShobanaEnglish R. KanakarathinamEnglishEthics, Value education, Need of ethics, Awareness of ethicsIn today’s goal driven society, youth are capable of generosity, solidarity and loyalty. They are less socially inferred and highly individualistic with their own ideas and values. They face numerous issues like unemployment, psychological instability, disruptive behaviour and numerous life problems. Code of ethics should be addressed to the youth by making awareness through education system. The good and bad cause on code of ethics should be addressed to the students to inculcate in their daily routine. Experts from academics and industries have stated that students should be trained and addressed regularly on their ethical behaviour and code of ethics thereby students will gain confidence to face all the situations in their life .It also gives a meaning and role for their activities which is performed by them. Teachers with a high level of moral professionalism have a deep obligation to help students learn and behave ethically which supports the students to have a sustained growth in their life. The major benefits of ethical students are they will be highly committed, taking responsibility, giving respect , high tolerance, non-discrimination, compassion, better understanding and good citizen. Hence, this paper attempts to analyse the need and awareness of the value education for students by the perception of the teachers in Pollachi region. EnglishEthics, Value education, Need of ethics, Awareness of ethicsIntroduction Ethics is being interrupted differently in different situation. It is a  standard of behavior that tell us how human beings ought to act in the many situations in which they find themselves-as friends, parents, children, citizens, businesspeople, teachers, professionals. Students being the part of the society and tomorrow’s nation’s builders, ethics should be inculcated among the students through educational framework. The teachers should orient all students to analyse and understand what is “good” and what is “bad” or what is “wrong” and what is “right” or what to do and what not to do with reference to day-to-day commitments. Commonly ethics is perceived as judging others, family values, outcomes, legal rights, or specialists in solving personal problems but ethics actually mean two aspect. The Most essential character to be present in a human is the ability to discern right from wrong, good from evil and propriety from impropriety and the commitment to do what is right, good and proper. Ethics is not just a topic to mull or debate, it entails action. Focusing on essentials of Ethics and Values The rapid changes in the technological world have posed many challenges in the global era. In this globalized mechanical commercial world, expecting the future generation with honesty and commitment is a night mare for the society. Today all the children have an immense power of observation and their feelings are deep-rooted. They always observe their parents at home and their teachers in school. They are highly sensitive. It is right time where parental care to the children is on the wane. Today media play a vital role in the life of children , the adverse effect on the media has made the Children to accept a  unrealistic approach in all the sphere of the life . In this context, there is now a growing demand in education to inculcate, nurture and develop values and ethics, particularly among the youth of the nation. Ethics lies at the core of the teaching profession. Tomorrow prospects of our country depend upon the moral values imparted to students during their college life. The role that teachers are expected to play in the lives of their students has undergone significant change in recent years. (Beavis, 2004)1 has stated that teachers are expected to be moral guides. This paper aims to report the awareness and need of ethics and values in education which supports to frame their inner behavior. Exist  REVIEW OF LITERATURE ‘Rauni Rosonen’(2007)2 has found that society of individuals are widely and deeply conscious of ethical questions and are studying them is the target and means of the ethical growth in teacher education. ‘William A.L. Anangisye, PhD (OCT 2010)3, the  paper focuses on the initiatives and challenges of promoting ethics in teacher training colleges. The study has concluded that without a welldefined education policy in favour of promoting teacher ethics education, efforts to that effect are doomed to fail. Rukhsana Zia(2007)4, have focus on “quality of education” has sharpened as people have become concerned about a perceived rise in materialism as standards of living have improved;. This paper tries to delineate the global normative aims of education as a model for assessing the composition of the teacher education curriculum in Pakistan. The paper accepts that ethics and values education is still in a formative stage. Gunnel Colnerud (2006)5, research on teacher ethics and the moral dimensions of teaching has contributed to extensive and valuable knowledge, which has sometimes led to constructive syntheses of positions finally he found there is a  relationship between moral education and professional ethics and what is morally significant in the teaching profession. (Gallagher, 2009, p.11)6. An increasing number of child research publications reflect on research practice and demonstrate the “problematic, messy and contested nature of ethical dilemmas” Objectives of the study To find the factors influenced as ethical values among teachers. To know the perception of faculties for the need of ethic in education curriculum. To analyse the values that transform the character of the students To know the values that influence the balanced life in future Research methodology To obtain information needed to fulfill the objectives of the research study, questionnaire was drafted. This questionnaire was circulated among faculties in randomly chosen in different department of colleges in Pollachi region. 10 arts and Science College. Completely filled in 204 questionnaires were analyzed for analyzing the above said objectives.    From the analysis it found that majority of the respondents 27% belong to both arts and engineering field, 26% of the respondents belong to science stream and 20% of the respondents belong to teacher training college. TABLE 4 Average Rating of perception of ethical values The ethical values varies based on the individual perception, it has a high relation between the ethics and the code of awareness. Teachers being the major role in transforming the students this analysis have been rated to know the perception of ethical values which is more essential for the students.  Perception of ethical values was analyzed based on the 27 factors, five point likert scale was used to rate the variable. Self esteem is most important factor which enforces one’s confidence level in performing all activities in better manner,  it was occupied the top position and Tolerance level which reduce one negative character excelling out occupies the least rank. The ranking of the factors are given in the following rank: Self esteem, Honor, Honesty, Environmental friendly , Responsibility, Equality of opportunity, Reasoned argument, Fairness, Trustworthiness, Humility, Freedom, Commitment , Respect, Truth, Reverence for Life, Human worth and dignity, Responsible citizenship, Social Coherence, Kindness, Devotion, Compassion, Open-mindedness, Objectivity, Patriotism, Benevolence, Respect for other's rights, Tolerance. From the analysis, the teachers have opined that students are not concerned about ethics in their daily practices which is a sign of danger for the future, the teachers have revealed that, they refrain from accepting any gift or favor that might impair or appear to influence their professional decisions or actions in their career, which reflects teachers are ethically aware what to do and what not to do, it is high time to give awareness of ethics in their professional career which reduce the issues in the society also. Teachers are in the position to teach ethics in their daily routine class inhibiting where ever possible, which make awareness among students to sustain their behavior ethically sound.     The above table reveals the source of ethical and moral values gained by teachers are through their routine practices, through spiritual means they learn ethical values, interaction with public and comments through public is also a media which educate the ethical and moral values among teachers, through their colleagues, higher official and sometimes students also gain the source of getting awareness on ethical practices, then occupies their family, experts in the academic area are also the source of getting aware of ethical practices. The opinion of the teachers is very much important in the role of student. According to the study the essential character that transform the values of the students was integrity  which occupies the first position, it make the students to be honest in all their duties with commitment and responsibility, then occupies the discipline which transform the student to have self control in them, next occupies the  cooperation , good temper and efficiency which improve the values of the students to  perform based on the situation and need. From the analysis, the respondents have revealed that students ‘behavioral values’ has highest influence in balancing their life in future,  personal and educational values has the second priority and spiritual values , social values and moral values occupies the consecutive position. FINDING Findings in this study indicated that Majority of the respondents 52% are male and 48% of the respondents are female and mostly the respondents domicile area is semi urban and the teachers are equally distributed ranging from 20% to 30% in arts, science, engineering college and teachers training institute. Perception of ethical values  was analyzed based on the 27 factors, with five point likert scale techniques where self esteem was rated as the most important factor which enforces one’s confidents level in performing the activities in better manner,  it was occupied the top position. The ranking of the factors are given in the following sequence :  Self-esteem, Honor, Honesty, Environmental friendly, Responsibility, Equality of opportunity, Reasoned argument, Fairness, Trustworthiness, Humility, Freedom, Commitment , Respect, Truth, Reverence for Life, Human worth and dignity, Responsible citizenship, Social Coherence, Kindness, Devotion, Compassion, Open-mindedness, Objectivity, Patriotism, Benevolence, Respect for other's rights, Tolerance. From the analysis, the teachers have opined that students are not concerned about ethics in their daily practices which is a sign of danger for the future, the teachers have revealed that, they refrain from accepting any gift or favor that might impair or appear to influence their professional decisions or actions in their career, which reflects teachers are ethically aware what to do and what not to do, it is high time to give awareness of ethics in their professional career which reduce the issues in the society also. Teachers are in the position to teach ethics in their daily routine class inhibiting where ever possible, which make awareness among students to sustain their behavior ethically sound.    The above table reveals the source of ethical and moral values gained by teachers are through their routine practices, through spiritual means they learn ethical values, interaction with public and comments through public is also a media which educate the ethical and moral values among repondents, through their colleagues, higher official and sometimes students also gain the source of getting awareness on ethical practices, then occupies their family, experts in the academic area are also the source of getting aware of ethical practices. The opinion of the teachers is very much important in the role of student. According to the study the essential character that transform the values of the students was integrity which occupies the first position, it make the students to be honest in all their duties with commitment and responsibility, then occupies the discipline which transform the student to have self control in them, next occupies the cooperation, good temper and efficiency which improve the values of the students to  perform based on the situation and need. From the analysis, the respondents have revealed that students ‘behavioral values’ has highest influence in balancing their life in future,  personal and educational values has the second priority and spiritual values , social values and moral values occupies the consecutive position.   Suggestion From the thought of Swami Vivekananda - “We must have life-building, man-making, character building-education.” In the present digital era, there is wide opportunity to make awareness of the moral values and ethics values among students. It is high time to take necessary action to inculcate moral and ethical values in their curriculum and have a practice of formal and informal discussion on daily routine in all aspects from personal to career. The following suggestions can be implemented Including moral and ethical values in their curriculum Providing value orientation in their curriculum than money orientation. Teaching through innovative methods to motivate them to get aware Conduction of programmes regularly stressing the values such as Personal Values, Social Values, Cultural Values, Spiritual Values, National values, Family values, Universal Values. Giving counseling to students based on their emotional behaviour. More community oriented activities should be arranged and social issues and their solutions should be addressed. Moral science subject should be introduced till higher education level. Teacher should be real initiator, inspirer  and guiding person Should arrange training to inculcate the values in their regular practice, control emotions, to be compassion and lovable, taking responsibility etc. Conclusion The Radhakrishnan Commission (1948-49) highlighted the importance and the need to unite spiritual training in the curriculum of educational institutions. The Mudaliar Commission (1952-53) stressed that student’s character and the behavior would depend on religious and moral instruction. The Ramamurthy Committee (1990) reviewed that the essential quality of education is, it must develop a set of values like love , compassion, social order based on truth and nonviolence and integrating the science with spirituality. Considering the fact that students spend a great part of their life with teachers and in turn teachers contribute significantly into overall 'quality of students lives’. There are numerous possibilities for teachers to influence the students to transform their attitude in good manner, and to feel that they can analyse their life style and behavioral attitude by understanding what is right, and deciding what they can commit for themselves and others, to create the kind of society they want to live in. In the light of the findings of the research, it is concluded that our faculty are aware of their responsibilities in shaping the moral and ethical values of student. Englishhttp://ijcrr.com/abstract.php?article_id=816http://ijcrr.com/article_html.php?did=816 Beavis, Adrian (2004) "Why Parents Choose Public or Private Schools," Research Developments: Vol. 12 , Article 3.  Rauni Rosonen’(2007) ,”‘The Ethical Nature of a Teacher's Work “,Business and Organization Ethics Network Vol. 2, No. 1. ‘William A.L. Anangisye, PhD (OCT 2010),” Promoting Teacher Ethics In Colleges Of Teacher Education In Tanzania: Practices And Challenges”, African Journal of Teachers Education Volume 1 No. 1.Pages 64 – 77. Rukhsana Zia(2007),“Values, Ethics and Teacher Education: A Perspective from Pakistan” Higher Education Management and Policy, vol. 19, issue 3, pages 20. Gunnel Colnerud (2006),” Teacher ethics as a research problem: syntheses achieved and new issues” Teachers and Teaching, Volume 12, Issue 3,pp  365-38 Gallagher, M. (2009). Ethics. In E.K. Tisdall, J. Davis & M. Gallagher (Eds.), Researching with children and young people: Research design, method and analysis. London: Sage Publications Code of Professional Ethics for School Teachers, National Council for Teacher Education, Wing – II, Hans Bhawan, 1, Bahadur Shah Zafar Marg, New Delhi- 110002 December, 2010 Roli Rai, Inculcation of Values: A Necessity Today. Int. J. Educat. Res. Technol. Vol 5 [1] March 2014. 30-32   Australian Journal of Teacher Education ,Vol 33, 4, August 2008 1”Making The Implicit Explicit: Values And Morals In Queensland Teacher Education”-Amanda Mergler , Queensland University of Technology Indian Education System: What needs to change? Unlawyered, National University of Juridical Sciences. Available at: http://startup.nujs.edu/blog/indian-education-system-what-needs-to-change/ [Accessed May 16, 2015] Significance of Ethics in Education by Justice J.S. Verma, http://www.ugc.ac.in/page/Other-Publications.aspx /[ Accessed Dec ,2017] Concerns of Value Education: Role & Responsibilities of Educational Institutions,http://www.qcin.org/nbqp/qualityindia/vol-2-no2/column.html/[ Nov 2011]  Indian Education System Needs A Change, http://www.indigenousherald.com/index.php/education/97-values-education-and-humanrights-challenges-and-suggestions/[July 2015] History of education in the Indian subcontinent, https://en.wikipedia.org/wiki/History_of_education_in_the_Indian_subcontinent/ [Mar 2017]  

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524199EnglishN2017May15TechnologyDiagnosing Inter Firm Profitability of Pharmaceutical Industry: An Empirical Analysis for India English3236Abhinna SrivastavaEnglishIndian pharmaceutical industry ranks 3rd in the world in terms of volume, quality and range of medicines it manufactures. Cost of producing medicine in India is considerably lower than that of the USA and approximately half of that of European countries which confers a competitive edge to India over others. Current domestic market of Indian pharmaceutical industry is worth US$13.8 billion (PWC, 2013). There are hundreds of pharmaceutical companies in India so a comparative study of their profitability is always needed. Present study is an effort to give an insight into profitability measures of selected pharmaceutical companies in India. Top five pharmaceutical companies in India have been selected for study based on the size of their current market capitalization including Sun Pharmaceuticals, Lupin, Dr. Reddys Lab, Cipla and Aurobindo Pharma. Further in order to draw a conclusion, profitability ratios of these companies has been analyzed and ranked on the basis of their composite performance during the period of study i.e. FY 2012 to FY 2016. Present study shows Indian pharmaceutical companies are doing well on account of profitability measures; Lupin is far ahead of its competitors whereas Sun Pharma emerged as the least performer during the study period. EnglishProfitability, Sun Pharmaceuticals, Lupin, Dr. Reddys Lab, Cipla, Aurobindo Pharma, IndiaINTRODUCTION Indian pharmaceutical sector plays a pivotal role in economy development. It contributes approximately 2.4% of the global pharmaceutical industry in terms of value and 10% in terms of volume. Pharmaceutical industry in India is expected to grow at a CAGR of 16% from US$ 20 billion in 2015 to US$ 55 billion by 2020. Indian drugs and medicines are currently exported to more than 200 countries worldwide. USA is the focal market for our pharmaceutical products. India is the largest producer of generic medicines in the world and it is expected to grow further in years to come. Government of India plans to set up a venture capital fund of US$ 640 million to facilitate drug discovery and reinforce pharmaceutical infrastructure in the country. The ‘Pharma Vision 2020’ by the Department of Pharmaceuticals, Government of India is determined to make India a hub for major drug discovery in the world. One of the important aspects with Indian pharmaceutical companies is its cost effective production, cost of producing medicine in India is considerably lower than that of the USA and approximately half of that of European countries which confers a competitive edge to India over others. Leading Pharmaceuticals companies in India on the basis of their market capitalization includes Sun Pharmaceuticals, Lupin, Dr. Reddys Lab, Cipla, Aurobindo Pharma etc. OBJECTIVE OF THE STUDY To evaluate the performance of leading pharmaceutical companies in India in respect of their profitability. To scrutinize the profitability measures of these companies under the study; To make to a comparative analysis of the performance of these companies based on key profitability ratio; and To give an ultimate ranking to these companies on the basis of their profitability. RESEARCH METHODOLOGY Sample Selection: For the purpose of study top five pharmaceutical companies in India have been selected on the basis of their market capitalization and listing on both the Indian stock exchanges; BSE & NSE. Source of Data: The present study is based on secondary data, compiled from annual reports of these companies as published on their official website. Apart from it relevant data have been taken from the site of Indian stock exchanges; BSE, NSE, financial sites like Money control and Google finance. Various financial journals, magazines and report have also been used for the present study. Further data have been scrutinized with the help of MS word, Excel etc. Methodology: For the purpose of study data so obtained was analyzed by using ratio analysis of favorite profitability measures like Operating Profit Margin, Net profit Margin, Return on Equity, Return on Capital Employed and Return on Assets. An ultimate ranking was given to each company on individual performance at each profitability ratio. Period of Study: The present study covers a period of five years i.e. FY’12 to FY’16. Scope of the Study: Present study encompasses only top five pharmaceutical companies; Sun Pharmaceuticals, Lupin, Dr. Reddys Lab, Cipla and Aurobindo Pharma based on their current market capitalization. This paper concludes on a single parameter i.e. profitability measure. DATA ANALYSIS For the purpose of study a list of top five pharmaceutical companies has been categorized on the basis of their market capitalization as on 15th September, 2016. Table-1 exhibits top five IT companies in BSE and their market cap in Rs. Cr. Based on five years average profitability measures a datasheet (Table-2) has been prepared for top five pharmaceutical companies. All the measure profitability ratios; Operating Margin, Net Profit Margin, Return on Net worth/Equity, Return on Capital Employed  and Return on Assets were calculated further mean for five years FY’12 to FY’16 in term of percentage have been taken for the study. 1. Operating Margin: Operating Profit Ratio constitutes the relationship between operating profit and net sales of the company. Operating Profit is also termed as PBIDT or EBIDTA; profit/earnings before interest, depreciation, tax and amortization. A higher operating margin ratio makes a company able to assure enough margins to cater its non-operating expenses. It also helps to create sufficient reserve to ensure payment of dividends. Operating Profit Ratio is computed as follow; Figure-1 exhibits that Lupin maintains higher average operating margin at 31.91% in last five years; FY 2012 to FY 2016, followed by Dr. Reddys Lab (26.26 %), Aurobindo Pharma (24.71 %) and Cipla (23.52%). Sun Pharma is maintaining least operating margin at average 17.13 %. 2. Net Profit Margin: Net Profit Ratio establishes the relationship between net profit and net sales of the company and shows managerial efficiency of the company in measure areas like manufacturing, administration and selling the product. Higher Net Profit margin shows a profitable position of the company which help them to endure in the case of increasing cost of production, deteriorating selling prices and declining demand for the product in the market. Net Profit Ratio is calculated as under: Figure-2 exhibits that Lupin maintains highest average Net Profit Margin at 21.75% in last five years; FY 2012 to FY 2016, followed by Dr. Reddys Lab (15.68%), Cipla (14.51%) and Aurobindo Pharma (9.54 %). Sun Pharma on the other hand has negative net profit margin at average -13.79 %. Return on Equity: For investors of the company Return on Equity (ROE) /Return on Net worth measures the return on the investment into the equity shares of the company, it is the most important profitability measure for an investor. The higher the Return on Equity ratio, the better it is supposed for the investors. ROE exhibits with some exceptions, firm capability of using investors' money in the most profitable manner. Return on Equity (ROE) computed as follow; ROE = Net Profit / Shareholders Fund ×100 Figure-3 exhibits that Lupin is having highest average ROE at 26.45% in last five years; FY 2012 to FY 2016, followed by Dr. Reddys Lab (15.60%), Aurobindo Pharma (15.30%) and Cipla (13.53%) while Sun Pharma is the least performer with ROE of 5.63%. 4. Return on Capital Employed: ROCE exhibits firm’s operational efficiency, it measures to what extend investors are getting return on the capital employed by them in the company. ROCE ratio is a performance measure which simply shows how much return is generated by the investors from their invested capital. The ROCE is computed as follow; Figure-4 exhibits that Lupin maintains highest average ROCE at 25.06% in last five years; FY 2012 to FY 2016, followed by Dr. Reddys Lab (14.39%), Cipla (12.98%) and Aurobindo Pharma (12.59%). Sun Pharma is the least performer among all the companies having negative ROCE at average -2.08 %. 5. Return on assets (ROA) Return on assets commonly known as ROA, ascertains the relationship between firm’s earning capacity and its long term assets. ROA reveals how beneficial a firm is towards its total long term assets and how efficiently the management is using its assets in order to breed maximum possible earnings. It is calculated as a percentage as follow; Figure-5 exhibits that Lupin maintains the highest ROA at average 19.88% in last five years; FY 2012 to FY 2016, followed by Cipla (10.57%), Dr. Reddys Lab (14.12%), and Aurobindo Pharma (9.98%). Sun Pharma even at ROA measure stood last with negative average of -0.67 %. DISCUSSION: Table-3 shows consolidated profitability measures of top five pharmaceutical companies in India. As per profitability ratio, firstly individual rank has been given to each company based on isolated profitability measure then an ultimate rank to all the five companies on the basis of their individual profitability rank. Highest rank has been given to the company having minimum total value of individual ranking in each profitability measure. FINDINGS: 1. It was found that Lupin among the top five pharmaceutical companies in India selected for the study is the top performer on all the profitability parameters. It maintains the highest operating profit margin (OPM %) at average 31.91%, net profit margin (NPM %) 21.75%, ROE 26.4%, ROCE 25.06% and ROA 19.88% on average basis from FY 2012 to FY 2016. 2. Dr. Reddys Lab stood the second position on all parameters except ROA. It’s average operating profit margin (26.26%) net profit margin (15.68%), ROE (15.6%) and ROCE (14.39%) were just behind that of Lupin while on ROA (10.12%) basis it was on third position. 3. Sun Pharmaceutical was the least performer on all the profitability parameters used in our study. It’s average OPM%, NPM%, ROE, ROCE and ROA were 17.13%, -13.79%, 5.63%, -2.08% and -0.67% respectively during FY2012 to FY 2016. 4. In the area of operating profit margin (OPM%), Lupin stood first at average 31.91% followed by Dr. Reddys (26.26%), Aurobindo Pharma (24.71%), Cipla (23.52%) and Sun Pharma (17.13%) during FY 2012 to FY 2016. 5. Among the top five leading Pharmaceutical companies in India Lupin maintains the highest net profit margin (NPM%) at average 21.75% followed by Dr. Reddys (15.68%), Cipla (14.51%), Aurobindo Pharma (9.54%), and Sun Pharma (-13.79%) during FY 2012 to FY 2016. 6. On return on equity (ROE) measure, Lupin was at the top at average 26.45% followed by Dr. Reddys (15.60%), Aurobindo Pharma (15.30%), Cipla (13.53%) and Sun Pharma (5.63%) during FY 2012 to FY 2016. 7. In the area of return on capital employed, Lupin maintains highest ROCE at average 25.06% among top five pharmaceutical companies in India while remaining companies Dr. Reddys (14.39%), Cipla (12.98%), Aurobindo Pharma (12.59%) and Sun Pharma (-2.08%) are far behind it. Thus Lupin is efficiently utilizing its capital. 8. During the period of study the highest return on assets (ROA) was observed in Lupin at average 19.88% followed by Cipla (10.57%), Dr. Reddys (10.12%), Aurobindo Pharma (9.98%) and Sun Pharma (-0.67%). CONCLUSION: Lupin among the top five Indian pharmaceutical companies selected for the study maintains highest score at every parameter of profitability analysis measures used. It secured 1st position based on ultimate ranking in our study with operating profit margin (OPM) at average 31.91%, Net profit margin 21.75%, ROE 26.4%, ROCE 25.06% and ROA 19.88% during last five years performance. On the other hand, Sun Pharma among selected pharmaceutical companies maintains least score at every parameter of profitability, It secured overall 5th position in our study based on ultimate ranking with operating profit margin (OPM) at average 17.13%, Net profit margin -13.71%, ROE 5.63%, ROCE -2.08% and ROA -0.67% during last five years performance. The current study shows Indian pharmaceutical companies selected for the study are doing well on account of profitability measures; however Sun Pharma could not deliver good numbers in recent years for which further study is required. Lupin posted outstanding numbers during last five years and is far ahead of its competitors by securing 1st rank in our study. Dr. Reddys Lab, Cipla and Aurobindo Pharma based on ultimate rank though far behind of Lupin secured 2nd, 3rd and 4th position respectively. However, the profitability performance of Sun Pharma with 5th ultimate rank has been posting consistently poor numbers among top five pharmaceutical companies in the India. ACKNOWLEDGEMENT Author acknowledges the immense help received from the scholars whose articles are cited and included in references of 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. Englishhttp://ijcrr.com/abstract.php?article_id=817http://ijcrr.com/article_html.php?did=817 Annual reports of Sun Pharmaceuticals, Lupin, Dr. Reddys Lab, Cipla and Aurobindo Pharma,for the year 2012 to 2016, retrieved from their official websites. Singh, Vineet and Srivastava, Abhinna, (2015) Receivables Management in Leading Heavy Electrical Industries in India, International Journal of Management, 6(4), 2015, pp. 1 - 8. F. Arditti, (1967), “Risk and the Required Return on Equity”, ‘The Journal of Finance’, VOL. 22, No. 1, p. 19-36. Vineet Singh. Significance of Working Capital Turnover Ratio: A Case Study of BHEL and Crompton Greaves, International Journal of Management, 6(3), 2015, pp. 1 – 7. G. Bharathi Kamath, (2008), "Intellectual capital and corporate performance in Indian pharmaceutical industry", Journal of Intellectual Capital, Vol. 9 Issue: 4, ISSN: 1469-1930, Emerald Group Publishing Limited, pp.684 – 704. Srivastava, Abhinna (2016), ‘Opportunities and Threats of Mega Merger: A Case Study of Sun Pharmaceuticals and Ranbaxy Laboratories’, International Journal of Commerce and Management Research, ISSN: 2455-1627, Volume 2; Issue 8; August 2016; Page No. 112-115. Srivastava, Abhinna (2016), ‘An Empirical Analysis on Inter Firm Profitability of IT Sector In India’, Abhinav National Monthly Refereed Journal of Research in Commerce & Management, Volume 5, Issue 10 (October, 2016), ISSN-2277-1166. ‘Annual reports’, Official website of NSE India; retrieved on 15th September, 2016 from; https://www.nseindia.com/ ‘Annual reports’, Official website of BSE India; retrieved on 15th September, 2016 from; www.bseindia.com/ Official website of Money n Business; retrieved on 20th September, 2016 from;  http://www.moneynbusiness.com/ ‘Pharmaceutical industry in India’: Retrieved on 25th September, 2016 from;  https://en.wikipedia.org/wiki/Pharmaceutical_industry_in_India ‘India Pharma Inc.: Capitalising on India’s Growth Potential’, Retrieved on 17th September, 2016 from; https://www.pwc.in/assets/pdfs/publications 2011/pwc_cii_pharma_summit_report_22nov.pdf ‘Indian Pharmaceutical Industries’: Retrieved on 15th September, 2016 from; http://www.ibef.org/industry/pharmaceutical-india.aspx IBEF 2016; Indian Pharmaceuticals Industry Analysis, retrieved on 15th September, 2016 from; http://www.ibef.org/industry/indian-pharmaceuticals-industry-analysis-presentation Moneycontrol financial; retrieved from;  http://www.moneycontrol.com/ Yahoo finance; retrieved on 15th September, 2016 from; https://in.finance.yahoo.com/

Radiance Research AcademyInternational Journal of Current Research and Review2231-21960975-524199EnglishN2017May15TechnologyStudy of Adventitious Lung Sounds of Paediatric Population using Artificial Neural Network Approach English3745Sibghatullah I. KhanEnglish Vasif AhmedEnglishObjectives: Human lung sounds are important indicators of underlying lung pathology. The prime objective of this work is to classify normal and adventitious lung sounds in paediatric population using spectral features and artificial neural networks. Material and Method: 3M Littmann 3200 electronic stethoscope was used to record the lung sounds. After pre-processing ten spectral features were extracted. For classification, comparative performance of different artificial neural networks is studied and GFF neural network with calculated optimum parameters is selected. Results: For testing data Out of 49 normal subjects 48 were classified successfully and out of 52 pathological subjects 48 were classified successfully. The classification sensitivity and specificity obtained is 92.30% and 97.95% respectively. Conclusion: Early diagnosis of lung disorder is important especially in childhood so that further progress of the disease could be prevented. New approach to detect adventitious lung sounds is being proposed utilizing electronic stethoscope as a recording device. Combination of spectral features and artificial neural networks has provided classification accuracy of 95.12%. EnglishLung disease, Adventitious lung sounds, Spectral features, Artificial neural networks INTRODUCTION Human lung sounds play an important role in diagnosis of underlying respiratory pathology. Traditionally medical doctors used to auscultate the lung sounds with the conventional stethoscope. This approach is quite common, but is subjected to some concerns like, for the novice doctors it is difficult to distinguish between different categories of adventitious lung sounds due to unavailability of any objective reference. Moreover, the misdiagnosed adventitious lung sounds of paediatric population may lead to adulthood repository disease such as COPD. Respiratory disorders, if diagnosed early in childhood, could be cured with proper antibiotics. To sum it up, it is very important to diagnose adventitious lung sounds in the paediatrics population due to their inability to communicate effectively about their health problems and lack of research in this category. This problem is more severe in the developing countries like India where majority (70%) of the population resides in the rural regions where appropriate medical facilities are not easily available due to distant geography and due to the shortage of trained medical practitioners.  There are estimated 6.4 million deaths [1] due to lung disease globally and in India it accounts for 11% of all the deaths [2]. Respiratory disorders are the second most cause of mortality in India after Heart related disease. The aim of this study is to address the issue by employing signal processing techniques in analysis of paediatric lung sounds to categorize them in normal and adventitious category. Now-a-days commercialization of electronic stethoscope with various inbuilt features opens a wide opportunity for the researchers in the biomedical field. Littmann is most trusted brand in the stethoscope market since many years. 3M Littmann traditional models were the most popular amongst the doctors especially amongst the pulmonologists and cardiologists. 3M Littmann 3200 Electronic stethoscope is used to record the lung sounds of subjects which was not utilized for lung sound recording and analysis purpose by previous researchers. The paper has been organized into four sections. The second section presents review of some research done in past. Section three describes materials and method which includes data acquisition, feature extraction and classification. Finally Section four presents results and fifth describes conclusion. LITERATURE REVIEW Table A provides some of the research work done in the past for automated objective analysis of lung sounds.   From table 1, it is noted that lot of research is done and still going on relating the automatic/computerized analysis of adventitious lung sounds and many signal processing techniques have been employed. The majority of the research carried out was for the adult population and vast non-uniformity is observed in the data acquisition methods, Some researchers have utilized lung sound training tapes where as some of them have used data from online resources. In spite of this, majority of the researchers have used the data which was acquired using microphones, microphones embedded with stethoscope or sensor jackets. There are in fact, very few researchers who have used data recorded from paediatric population. Also no one till date has utilized the electronic stethoscope model 3200 by 3M Littmann with the feature of ambient noise reduction. So there exists tremendous scope to study the lung sounds of paediatric population for objective analysis and for classification.    MATARIALS AND METHODS A] Data Acquisition For data acquisition, 3M Littmann 3200 electronic stethoscope was used .3M Littmann electronic stethoscope is been chosen because of its ambient noise reduction capability, larger bandwidth (0-2000 Hz) and facility to record in extended mode which provides maximum bandwidth as compared to traditional stethoscope which provides only two modes i.e. Bell and Diaphragm. Also 3M Littmann is most popular and trusted brand among medical practitioners. Also its fidelity for heart sound recording has been tested in our precious study [32]. The stethoscope has the Bluetooth connectivity with PC with the help of ‘StethAssist’ software provided by Littmann. One complete recoding of 60 sec is transmitted to PC simultaneously during auscultation through Bluetooth interface. The file is then exported in ‘.wav’ format (16 bit PCM sampled at 4000 Hz). Recordings were made on PC running Windows 8 operating system with AMD quad core processor with 4 GB ram. The recording setup is shown in figure 1. All lung sound recordings were made after obtaining approval from concerned authorities and in accordance with medical ethics. The mean age of children selected for recording is 2 years ± 1 year, with almost equal gender distribution. The recordings were carried out at renowned child hospitals having Paediatrician with more than 15 years of experience in Nagpur city and in the towns of Pusad and Digras (Vidharbha region of Maharashtra State India). B] Sorting and Pre-processing The recordings were labelled according to the disease and sorted according to the quality. Here quality means the absence of hospital noise. The noise in the data was mainly due to crying and movement of child subjects. So after sorting, almost 50% of the data was discarded due to high amount of noise content. Total 540 recording were recorded out of which only 253 recording were selected of which 127 include adventitious lung sounds such as wheezing, crackles, grunting, crepitations and harsh breath sounds. For categorizing normal lung sounds, 126 recordings were selected including the lung sounds of the subjects having common acute cough and cold. The pre-processing stage involves filtering, DC removal, segmentation and normalization. Below 100 Hz the auscultation sounds are dominated by heart sounds, it is necessary to remove this unwanted signals by means of suitable filtering which will not only remove the noise but will also preserve the basic nature of lung sounds for higher frequencies. Different digital filters were designed and tested by varying order of filter and window types, and finally 7th order Chebyshev type I IIR filter with cut-off frequency of 100 Hz is selected after observing time and frequency domain characteristics of filtered signal. Filter designing is done using MATLAB 2008b licensed version. After successful filtering of all the recordings, two breath cycles from each recording is extracted manually using WAVEDIT software. Each segmented recording is relabelled and saved for analysis. Segmented sounds are then amplitude normalized in the range of ±1.These pre-processed sounds was then used for feature extraction and classification. C] Feature Extraction: By observing time and frequency domain description (spectrum) of all the cases of normal and adventitious lung sounds, it was observed that time domain analysis would not help good classification because of similarity in shape of time domain statistical parameters. The previous studies related to time domain analysis of lung sounds also do not reflect encouraging results in terms of accuracy. So, frequency domain analysis is carried out using spectral features which were extensively used in automatic speech recognition systems [33-34]. Total ten spectral features were extracted consisting of Spectral centroid, Spectral crest, Spectral decrease, Spectral flatness, Spectral flux, Spectral roll off, Spectral skewness, Spectral kurtosis, Spectral Slope and Spectral spread. Brief definition of spectral features is given below Spectral Centroid It is the centre of gravity of spectrum. It is defined as Spectral centroid basically represents the location of concentration of spectral energy. Low values of spectral centroid indicate presence of lower frequency components and vice versa. 2.Spectral Crest Factor It is a measure of tonalness of the signal. It is the ratio of maximum of the magnitude spectrum to the sum of all bins in the magnitude spectrum. It is defined as Where n is the block length. In this study the complete segmented signal (two breath cycles) were considered as single block. Low values of spectral crest factor indicates flat spectrum where as high values indicates a sinusoidal. Spectral crest factor is zero for the blocks having zero energy (silence). 3.Spectral Decrease The spectral decrease measures the steepness of the decrease in the spectral envelope over frequency. It is defined as The value of spectral decrease is a value S.Dec≤1. A lower value of spectral decrease indicates concentration of the spectral energy at bin 0. Spectral Flatness The spectral flatness is the ratio of geometric mean and arithmetic mean of the magnitude spectrum, it is defined as The value of spectral flatness is greater than 0.The upper value depends on the maximum spectral magnitude. Non-flat spectrum tends to have lower values of S.F where as flat spectrums results in higher values of S.F. Spectral Flux The spectral flux measures the amount of change in spectral shape. It is defined as the average difference between successive STFT frames The value of spectral flux lies in the range 0 ≤ S.F ≤ A with A representing maximum possible spectral magnitude. Low values of A represents steady input signals. Spectral Kurtosis The Kurtosis is referred to the ratio of   fourth central movement of a variable to the fourth power of standard deviation. The spectral kurtosis measures how much the distribution of spectral magnitude resembles the Gaussian distribution. It is defined as Spectral kurtosis represents peakedness of the signal. For spectrum having peaks, its value will be higher. Again one complete segmented signal is used as input to calculate spectral kurtosis. Spectral Roll off It is the measure of concentration of spectrum. It is defined as the frequency below which certain percentage (In this study 95%) of the magnitude distribution of the spectrum is concentrated. If mth DFT coefficient corresponds to the spectral roll off of the ith frame then C is the adapted percentage which is 95% in our case. To normalize spectral roll off frequency it is divided by the FL (total length of the band). So it will have values between 0 and 1, where 1 corresponds to the maximum frequency of signal (fs/2).This parameter actually describes the distribution and shape of the spectrum i.e. narrower spectrum yields lower values where as wider spectrum results in higher values of spectral roll off. Spectral Skewness The Spectral skewness is a measure of symmetry of distribution of the spectral magnitude around their arithmetic mean. It is defined as It indicates the amount of non similarities between spectral magnitudes i.e. for flat like spectrums it has a very low value, where as for fluctuating spectrum its value is high.   Spectral Slope       The spectral slope is similar to the spectral decrease which measures the slope of the spectral shape. It is calculated using a linear approximation of the magnitude spectrum. In the presented form, the linear function is modelled from the magnitude spectrum. It is calculated with the following equation     Spectral Spread It is defined as the second central moment of the spectrum. To calculate it deviation from the spectral centroid is taken   To normalize SS in the range [0,1], it is divided by the factor (fs/2) ,where 1 corresponds to maximum frequency of signal (fs/2). All ten spectral features are calculated for each segmented recording. The calculations were performed in Matlab R2008b Licensed version using toolbox available [34]. For 126 normal subjects the dimension of feature matrix is 126*10 and for 127 Pathological subjects the feature matrix dimension is 127*10. By observing both the feature matrices it is concluded that they are not linearly separable, so classification based of artificial neural network has been employed. C] Classification: Neurosolutions (Neuro Dimension Inc.USA) 5.07 was used to implement different NN based classifiers on lung sound recordings which are represented by feature vector containing 10 different elements. This paper explores the Multilayer Perceptron (MLP), Generalized feed forward (GFF) and Modular Neural networks for classification purpose. In the first stage all the network classifiers were tested for different topologies i.e. by varying number of hidden layers and processing elements (PEs) and by changing transfer function and learning rules. Performance measures such as mean square error (MSE), minimum absolute error, maximum absolute error, mean absolute error (MAE), correlation coefficient and classification accuracy were calculated for different classifiers. After observing performances of all the possible combination of transfer functions and learning rules with varying processing elements in hidden layers, it was decided to use network having one hidden layer with six processing elements in it. Table I provides comparative performances in terms of minimum MSE for MLP, GFF and MNN, where as table II provides their performance in terms of minimum absolute error, maximum absolute error, correlation coefficient and classification accuracy on testing data. From tables I and II it is concluded that GFF outperforms MLP and MNN in terms of performance measures. The classification accuracy attained by GFF for normal and adventitious lung sound is 90.90% and 91.22% respectively. So in order to optimize GFF for improved accuracy, network is trained by different combinations of transfer functions and learning rules. Table III and IV illustrates the comparative performance of the network for various combinations of transfer functions and learning rule From the table III it is observed that tanh, which is a non linear transfer function performs better than other transfer functions. This might be due to high degree of non linear separability in the data. So in the next stage of network development, different learning rules such as Step, Momentum, Conjugate gradient (CG), Levenberg Marquardt (LM), Quickprop (QP), Delta bar delta (DBD) were used along with Tanh as a transfer function. Table IV shows comparative performances in terms of Mean square error (MSE), Mean absolute error (MAE), Correlation coefficient  and classification accuracy for different learning rules. Maximum classification accuracy and correlation coefficient value was obtained by the tanh- CG combination. The computed value of MSE and MAE is also minimum for this combination. All the performances were calculated for testing data. From Table III and IV it is evident that GFF with tanh as transfer function with Conjugate gradient as a learning rule performs better than other transfer function-learning rule combinations. The final specifications of the network after carrying out trials can now be defined as follows:   Network: Generalized feed forward NN Stopping condition: 3000 Epochs Conscience rule: L2 Norm Number of hidden layer: 01 Number of PEs in hidden layer: 06 Hidden Layer Transfer function: Tanh           Hidden layer learning rule: Conjugate gradient Output layer: Transfer function: Tanh Output layer Learning Rule: Conjugate gradient                                                                                                                                                                                              RESULTS Table IV shows the results using GFF neural network in terms of performance measures such as MSE, MAE, Correlation coefficients and classification accuracy. The sensitivity of neural network for all the ten features is shown in figure 2. For testing data of 49 subjects belonging to normal category, 48 were classified successfully and for Subjects belonging to pathological category out of 52, 48 were successfully classified. The sensitivity and specificity for conducted study is 92.30% and 97.95% respectively leading to the overall accuracy of 95.12%. CONCLUSIONS A new approach to preliminary detect adventitious lung sounds in paediatric population has been proposed in this study. Features representing spectral characteristics were calculated for each recording and subsequently different artificial neural network topologies involving MLP, GFF and MNN have been tested by varying number of hidden layers, PEs, transfer functions and learning rules. It has been concluded that GFF with one hidden layer and with 6 PEs in it incorporating tanh as transfer function with Conjugate gradient as learning rule is the most optimum neural network for this application. The overall accuracy obtained is 95.12%. The work can be further extended to classify different categories of adventitious lung sounds for detecting specific disease after suitable increase in the subjects recordings pertaining to each disease. ACKNOWLEDGEMENTS We acknowledge the valuable support received from Shri J. S. Naik, President Janata Shikshan Prasarak Mandal,  Pusad, Dr. H. B. Nanvala, Principal Babasaheb Naik College of  Engineering Pusad and  Dr. N. P. Jawarkar, Head Department of Electronics and Telecommunication Engineering B. N. College of Engg. Pusad. We also convey our thanks to Dr. N. A. Charniya, Dr. S. N. Dandare and Prof Vijay Agrawal  for their help and support during this study. We also thank Dr. Mohibul Haque (Paediatrician, Nagpur), Dr. Arif Ahmed  (Paediatrician, Pusad), Dr. V.K. Deshpande  (Paediatrician,  Digras), Dr. Adanaul Haque Khan and Dr Anwar Siddiqui Nagpur for their help, guidance and support during auscultation recording of lung sounds. 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: None Conflict of Interest: None Abbreviation Chronic Obstructive Pulmonary Disease (COPD) Infinite Impulse Response filter (IIR-Filter) Spectral centroid (SC) Spectral crest factor (SCr) Spectral decrease (S.Dec) Spectral flatness (SF) Spectral flux (SFx) Spectral roll off (S.Roff) Spectral skewness (S.Skw) Spectral kurtosis (SK)                                                                                                                                                    Englishhttp://ijcrr.com/abstract.php?article_id=818http://ijcrr.com/article_html.php?did=8181. http://www.who.int/mediacentre/factsheets/fs310/en/ 2. https://www.cdc.gov/globalhealth/countries/india/pdf/india_ factsheet.pdf 3. K. J. Hong, W. Ser and D. C. G. Foo, “An analysis of sounds for lungs with excessive water,” 2016 38th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC), Orlando, FL, 2016, pp.3662-3665.doi: 10.1109/EMBC.2016.7591522 4. A. 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