Thrombolytic Potential of Aqueous and Methanolic Crude Extracts of Camellia sinensis (Green Tea): In vitro study

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ISSN 2278- 4136 ZDB-Number: 2668735-5 IC Journal No: 8192 Volume 2 Issue 1 Online Available at www.phytojournal.com Journal of Pharmacognosy and Phytochemistry Thrombolytic Potential of Aqueous and Methanolic Crude Extracts of Camellia sinensis (Green Tea): In vitro study Sikandar Khan Sherwani1*, Asma Bashir2, Syeda Sadaf Haider3, Muhammad Ajmal Shah4, Shahana Urooj Kazmi3 1. 2. 3. 4. Department of Microbiology, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan. [E-mail: sikander_biology@hotmail.com, Tel: +92-3245189042] Department of Biosciences, SZABIST, Karachi, Pakistan. Department of Microbiology, University of Karachi, Karachi, Pakistan. Department of Pharmacognosy, Federal Urdu University of Arts, Science and Technology, Karachi, Pakistan. Investigation of thrombolytic activity of crude extracts of Camellia sinensis (Green tea) using in-vitro thrombolytic model which is very simple, rapid and easy to do method. Both aqueous crude and methanolic extracts were studied however; the results of methanolic extract were more promising which exhibited maximum 95. 24% clot lysis as compared to aqueous one i.e. 90.34% at 800 μg/ml concentration in 72 hrs of incubation. Various concentrations of leaf extract i.e. 200μg/ml, 400μg/ml, 600ug/ml and 800μg/ml were tested at different time intervals including; 24hrs, 48hrs and 72hrs duration of incubation at 370 C for observing maximum clot lysis. The result indicated that concentrations of leaf extract enhanced the percentage of clot lysis in dose dependent manner. On the other hand, Streptokinase SK, a reference standard and water were used as a positive and negative control showed clot lysis maximum 96.63% and 41.32% in 72 hrs of incubation respectively. From results, it can be concluded that if further studies reveals the exact molecule from green tea diverse composition, an effective thromolytic candidate can be achieved for the improvement of the patients suffering from Atherothrombotic diseases. Keyword: Camellia sinensis, Thrombolytic Activity, Green Tea, Atherothrombosis. 1. Introduction Homeostasis maintains the integrity of circulatory system after damaging of the vascular channel[1]. Thrombus development is a critical event in the arterial diseases associated with myocardial infarction, anoxia, hypertension[2], stroke, and venous thromboembolic disorders that account for considerable number of deaths worldwide[3]. Currently all over the world; thrombolytic drugs like tissue plasminogen activator (t-PA), urokinase, alteplase, streptokinase etc. play a very important role in the management of patients with cerebral venous sinus thrombosis (CVST), is a common disorder that is often accompanied by Vol. 2 No. 1 2013 critical morbidity and mortality[4-7]. Urokinase (UK), streptokinase (SK) are cheaply available but their use is not as such safe due to its immunogenicity factor (Jennings, 1996), high risk of hemorrhage, anaphylactic reaction and lack of specificity[1,8]. Because of the shortcomings in the existing thrombolytic agents, a number of researches are underway to improve the variants of these drugs for their better effective nature[9]. Camiellia sinensis (Green tea) has been consumed for centuries as a hot beverage and has got immense medicinal properties[10]. The plant of Camiellia sinensis is an inhabitant of Southeast Asian region www.phytojournal.com Page | 125 Journal of Pharmacognosy and Phytochemistry commonly known as green tea[11]. Since long time, green tea is being widely consumed in China, Pakistan, India, North Africa and Middle East12. Green tea has been shown to have a wide range of beneficial physiological and [13] pharmacological effects . Moreover; studies indicated that green tea possess significant antioxidant, anti-inflammatory, anti-carcinogenic, antiviral, anti-adhesive to cell surfaces, antiprotozoal, probiotic, anthelminthic, neuroprotective antimicrobial[11,14-20]. The purpose of the undertaken research study was to find out the thrombolytic potential of Camiellia sinensis (Green tea) by in vitro-method. 2. Material and methods 2.1 Preparation of aqueous extract: The Green tea was purchased from the retail market in Karachi-Pakistan in reasonable price in large quantity were brought to the laboratory of Federal Urdu University of Arts, science and Technology (FUUAST)-Karachi-Pakistan for performing various biological activities. The extract was prepared in the concentration of 5% in distilled water. The aqueous extract was prepared in by boiling method of tea in water bath by constant agitation of for 15 minutes. Later, all the coarse suspended particles of tea were first removed by using strainer and then by passing via 0.22um filter (Sherwani et al., 2013). The extract was stored in refrigerator in small vials as aliquots till use. 2.2 Methanol Extraction: The methanol extractions of the active ingredient of the leaves were carried out using Harbone method[21]. 25g of the grinded leaves were soxhlet extracted using 250ml of 95% methanol. The extraction lasted for six hours. The volatile oil obtained was concentrated by evaporation using water bath at 100°C. 2.3 In vitro Thrombolytic analysis: The thrombolytic activity of Camiellia sinensis (green tea) was done by following the methods of using streptokinase (SK) as a standard reference[22-23]. Vol. 2 No. 1 2013 2.4 Streptokinase (SK) Solution Preparation: Commercially available lyophilized (Streptokinase) vial of 15, 00,000 I.U., was collected and then 5 ml of sterile distilled water was added and mixed properly. This suspension served as a stock from which 100μl (30,000 I.U) was used for in vitro thrombolysis[24-25]. Table 1: In vitro-thrombolytic activity of control (water) and standard (Streptokinase): Incubation time Clot lysis Standard Control 24hrs 48hrs 72hrs 24hrs 48hrs 72hrs 79.32% 92.22% 96.63% 8.85% 27.65% 41.32% Table 2: In vitro-thrombolytic activity of aqueous extract of Camiellia sinensis (Green tea): Concentrations of Incubation time Clot lysis crude leaf extract 24hrs 8.37% 200ug/ml 48hrs 24.44% 72hrs 42.72% 24hrs 12.55% 400ug/ml 48hrs 29.37% 72hrs 56.57% 24hrs 16.54% 600ug/ml 48hrs 43.68% 72hrs 70.84% 24hrs 21.73% 800ug/ml 48hrs 55.99% 72hrs 90.34% Table 3: In vitro-thrombolytic activity of methanolic extract of Camiellia sinensis (Green tea): Concentrations of Incubation time Clot lysis crude leaf extract 200ug/ml 24hrs 10.77% 48hrs 29.63% 72hrs 47.42% 400ug/ml 24hrs 16.95% 48hrs 36.77% 72hrs 61.77% 600ug/ml 24hrs 21.24% 48hrs 53.65% 72hrs 77.34% 800ug/ml 24hrs 26.33% 48hrs 67.79% 72hrs 95.24% www.phytojournal.com Page | 126 Journal of Pharmacognosy and Phytochemistry 2.5 Blood Collection: Whole blood (4 ml) was drawn from healthy human volunteers (n = 10) by phlebotomist without a history of oral contraceptive or anticoagulant therapy[26,24]. 500 μl of blood was transferred to each of the ten previously weighed alpine tubes to form clots. 2.5 Bioassay: 2.5.1 Effect of crude leaf extract on clot lysis: Blood sample (500μl) was distributed in pre weighed sterile micro centrifuge tubes and incubated at 370C for 90min for clot formation. After clot formation, the serum was finely and completely aspirated without disturbing the clot and the tubes were again weighed to determine the clot weight[ 27]: (Clot weight = Weight of the tube containing clot – Weight of the empty tube). Each eppendorf tube containing clot was properly labeled and 100 μl of plant extract was added to the tubes. On the other hand, as a positive control, 100 μl of SK and as a negative non thrombolytic control, 100 μl of distilled water were separately added to the numbered control tubes. All the tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, fluid obtained was removed and tubes were again weighed to observe the difference in weight after clot disruption[28]. Difference obtained in weight taken before and after clot lysis was denoted as percentage of clot lysis[29]. % of clot lysis = (wt of released clot /clot wt) × 100 2.5.2 Maximum clot lysis observation with respect to concentration and incubation of time Various concentrations of leaf crude extract of Camiellia sinensis (green tea) i.e. 200μg/ml, 400μg/ml, 600ug/ml and 800μg/ml were tested at various time intervals including; 24hrs, 48hrs and 72hrs duration of incubation at 370C for maximum clot lysis[28]. Vol. 2 No. 1 2013 3. Results and discussion Tea has a wide range of water soluble phytoconstituents[30]. Almost all the available thrombolytic agents still have significant shortcomings[31]. According to one of the reports, approximately, 30% of the pharmaceuticals are prepared from plants worldwide32 and are considered to be less toxic and freer from side effects than the synthetic one33. Wonderful efforts have also been carried in recent past towards the exploration, discovery, designing and development of natural pro-ducts with antiplatelet[34], anticoagulant[35], antithrombotic[36] and thrombolytic activity of the plants[26]. As from the research findings of the undertaken study, it was clear concentrations of leaf extract enhanced the clot lysis process in an order of increasing dose along with the incubation time factor. Moreover, both nature of crude extracts aqueous and methanolic exhibited thrombolytic activity; nonetheless much significant results were obtained in methanolic extracts that pointed out the fact the treatment nature of extract preparation release different bioactive compounds. A study indicated that Camiellia sinensis (Green tea) contains flavonoids and some other related members like aflavins and the arubigins[37]. Other than flavonoids, green tea also possesses a rich amount of with cathechins38 and poly phenols[39] that are expected to have biological activities. Some studies also pointed that as green tea is not fermented during processing preserving the enzymes and olive green color; on the contrary, black tea is fermented before drying. Fermentation can destroy some of the active components of black tea[40]. From our study, in case of aqueous Camellia sinensis (green tea) leaf extract maximum 90.34% clot lysis was achieved at 800 ug/ml concentration in 72 hrs of incubation as mentioned in Table No-2 while; methanolic extract had greater 90.24% clot lysis at 800 ug/ml concentration in 72 hrs of incubation as mentioned in Table No-3. However; streptokinase SK a reference standard and water were used as a positive and negative control that showed clot lyiss maximum 96.63% and 41.32% in 72 hrs of incubation respectively as indicated in Table No- www.phytojournal.com Page | 127 Journal of Pharmacognosy and Phytochemistry 1. A number of studies in past have shown that green tea impairs blood clotting in man41 as well in animals[42]. Similarly, other plants that also have thrombolytic activity that could be due to the wide range of composition like phytoconstituents including rich sources of alkaloids, flavonoids, tannins and terpenoids[43]. Not only plants, some reports like marine algae having a product called Seanol (phlorotannin – active compound), possessing the ability in promotion of dissolution of intravascular blood clot via antiplasmin inhibition[44]. Another remarkable achievement has been conducted in which thrombolytic agent fucoidan, a branched sulfated fucan extracted from brown seaweeds, having antithrombotic potential[45]. 4. Conclusion In conclusion, as literature that is already rich with beneficial effects of Camiellia sinensis (Green tea), an endeavor in our study to exploit the potential as thrombolytic agent was promising especially the methanolic version of tea extract. Further study ought to be conducted for the exploration of isolated molecule that can be effective, safer, cheaper, nontoxic enough for ameliorating the thrombosis conditions. 5. Acknowledgement The authors are highly thankful to Ms. Asma Bashir, Lecturer-Biosciences-SZABIST-KarachiPakistan for the providing StreptokinaseReference standard vial for conducting this study. 2. 3. Islam MA, Mahmud ZA, Rahman SMA, Md. Monirujjaman and Saha SK et al. Evaluation of Thrombolytic activity and Brine Shrimp Lethality Bioassay of Methanol extract of stems of Tinospora crispa. International Journal of Pharmaceutical Sciences and Research 2013; 4(3): 1148-1153. Sultana I, Noor MA, Barua J, Mahmood A, Das MC, Ibrahim MM, Chowdhury MM et al. In-vitro antiatherothrombosis activity of four Bangladeshi plants. International Journal of Green Pharmacy 2012; 6(1): 5-8. Furie B and Furie BC. Mechanisms of thrombus formation. New England Journal of Medicine 2008; 359(9): 938-49. Vol. 2 No. 1 2013 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 6. Reference 1. 4. 16. 17. 18. Allroggen H, Abbott RJ. Cerebral venous sinus Thrombosis. Postgraduate Medical Journal 2000; 76: 12-15 Watson RD, Chin BS, Lip GY et al. Antithrombotic therapy in acute coronary syndrome. British Medical Journal 2002; 352: 1348-13514. Gesler WM. Therapeutic landscapes: medical issues in light of the new cultural geography. Social Science & Medicine 1992; 34: 735–746. Collen D. Coronary thrombolysis: streptokinase or recombinant tissue-type plasminogen activator Annals Internal Medicine 1990; 112: 529–538 Anwar S.M, Khan.I.N, Sarkar M.M, Barua.S, Kamal .A.T.M, Hosen .M.Z. Thrombolytic & Cytotoxic Effect Of Different Herbal Extracts. IJPSR, 2011; Vol. 2(12): 3118-3121 Marder VJ. Recombinant streptokinase – opportunity for an improved agent. Blood Coagul Fibrinolysis. 1993 ; 4:1039 –1040. Mbata. T. I, Debiao. L, Saikia. A: Antibacterial Activity of The Crude Extract Of Chinese Green Tea (Camellia Sinensis) On Listeria Monocytogenes. The Internet Journal of Microbiology. 2006 Volume 2 Number 2. Sherwani SK , Khan M. M , Munir S., Shah M.A., Kazmi S.U. Anthelminthic potential of crude extract of Camellia sinensis (Green tea).International Research Journal of Pharmacy( 2013) (Accepted). Katiyar SK, Mukhtar H. Tea in chemoprevention of cancer: epidemiologic and experimental studies. Int J Oncol 1996;8:221–38. Hamilton-Miller J.M.T. Antimicrobial Properties of Tea (Camellia sinensis L.). Antimicrobial Agents and Chemotherapy, Nov. 1995, p. 2375–2377 Vol. 39, No. 11. Choi, Y.B.; Kim, Y.I.; Lee, K.S.; Kim, B.S; and Kim, DJ. (2004). Protective effect of epigallocatechin gallate on brain damage after transient middle cerebral artery occlusion in rats. Brain Res; 1019(1–2):47– 54. Bakkir K. L, Yassen R. T, Mustaffa R. M. In Vitro and In Vivo Study Of Green and Black Tea Antimicrobial Activity On Methicillin Resistant Staphylococus aureus. Bas.J.Vet.Res.Vol.10, No.2, 2011. Wakayama M, Suzuki K, Toda M, Okubo S, Hara Y and T Shimamura. Inhibition of the infectivity of influenza virus by tea polyphenols. Antiviral Res. 1993; 1: 289 - 299. – Lee J. H., Shim J. S., Chung M., Lim. S Kim. K. H. In vitro anti-adhesive activity of green tea extract against pathogen adhesion. Phytotherapy Research. Volume 23, Issue 4, 460–466 (2009). Bokuchava, M. A.and Skobeleva. N. I. 1980. The biochemistry and technology of tea manufacture. Crit. Rev. Food Sci. Nutr. 12:303–370. www.phytojournal.com Page | 128 Journal of Pharmacognosy and Phytochemistry 19. Kakuda, T. (2002). Neuroprotective effects of the green tea components theanine and catechins. Biol Pharm Bull; 25(12):1513–1518. 20. Toda M, Okubo S, Hiyoshi R, Shimamura T. The bactericidal activity of tea and coffee. Lett Appi Microbiol. 1989; 8: 123-5. 21. Harbone NV. Phytochemical method. A guide to modern techniques of plant analysis 2nd ed,Chapman and Hall London1994. p. 425. 22. Prasad S, Kashyap RS, Deopujari JY, Purohit HJ,Taori GM, Daginawala HF, Effect of Fagonia Arabica (Dhamasa) on in vitrothrombolysis, BMC Complementary and Alternative Medicine, 7(36), 2007, 1-6. 23. Daginawala HF, Prasad S, Kashyap RS, Deopujari JY, Purohit HJ and Taori GM. Development of an in vitro model to studyclot lysis activity of thrombolytic drugs. Thrombosis Journal, 2006, 4:14. 24. Kawsar.M.H, Sikder.M. A, Rana.M.S. Nimmi.I, Rashid.M.A. Studies of Thrombolytic, Antioxidant and Cytotoxic Properties of Two Asteraceous Plants of Bangladesh. Bangladesh Pharmaceutical Journal Vol. 14,No. 2, July 2011-4606- 103 25. Chowdhury SN, Badrul AM, Haque ASMT, Zahan R, Mazumder ME and Haque ME, In vitro Free Radical Scavenging and Thrombolytic Activities of Bangladeshi Aquatic Plant Aponogeton undulatus Roxb, Global Journal of Pharmacology, 5(1) , 2011, 27-32. 26. Rahman.M.A, Sultana.R, Emran. T, Saiful Islam.M, Ashiqur Rahman.M, Joti Sankhar Chakma.J.S, Rashid. H and Monirul Hasan. C.M. Effects of organic extracts of six Bangladeshi plants on in vitro thrombolysis and cytotoxicity. BMC Complementary and Alternative Medicine. 2013, 13:25 27. In vitro studies on antimicrobial and thrombolytic Activity of Swietenia Macrophylla King. Mallik. J, Banik R.K. Journal of Pharmaceutical Research and Opinion 2: 5 (2012) 45 – 48 28. Elumalai A, Eswariah CM, Chowdary V, Kumar R, Anusha M, Naresh K, Screening of Thrombolytic Activity of Bougainvillea glabra Leaves Extract by In-Vitro, Asian Journal of Research in Pharmaceutical Sciences, 2(4), 2012, 134-136. 29. Shahriar.M. (2013). Phytochemical screenings and Thrombolytic activity of the Leaf extracts of Adhatoda Vasic. The Experiment.Vol. .7(4), 438-441 30. Balentine D.A., Wiseman S.A. & Bouwens C.M. (1997). the chemistry of tea flavonoids. Critical Reviews in Food Science and Nutrition 37(8): 693704. 31. Nicolini FA, Nichols WW, Mehta JL, Saldeen TG, Schofield R, Ross M, et al. Sustained reflow in dogs with coronary thrombosis with K2P, a novel mutant Vol. 2 No. 1 2013 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. of tissue plasminogen activator. J Am Coll Cardiol. 1992; 20:228-235 Anwar AK, Ashfaq M, Nasveen MA: Pakistan Forest Institute, Peshawar NWFP. Pakistan; 1979:15 -35 Annapurna A, Kanaka, Mahalakshmi D, Murali KK. Antidiabetic equilibrium value of MeCpG steps (,+14 deg.) [31,44]. In comparison, methylation has a significantly lower stability cost when happening at major groove positions, such as 211 and 21 base pair from dyad (mutations 9 and 12), where the roll of the nucleosome bound conformation (+10 deg.) is more compatible with the equilibrium geometry of MeCpG steps. The nucleosome destabilizing effect of cytosine methylation increases with the number of methylated cytosines, following the same position dependence as the single methylations. The multiple-methylation case reveals that each major groove meth- PLOS Computational Biology | www.ploscompbiol.org 3 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning ylation destabilizes the nucleosome by around 1 kJ/mol (close to the average estimate of 2 kJ/mol obtained for from individual methylation studies), while each minor groove methylation destabilizes it by up to 5 kJ/mol (average free energy as single mutation is around 6 kJ/mol). This energetic position-dependence is the reverse of what was observed in a recent FRET/SAXS study [30]. The differences can be attributed to the use of different ionic conditions and different sequences: a modified Widom-601 sequence of 157 bp, which already contains multiple CpG steps in mixed orientations, and which could assume different positioning due to the introduction of new CpG steps and by effect of the methylation. The analysis of our trajectories reveals a larger root mean square deviation (RMSD) and fluctuation (RMSF; see Figures S2– S3 in Text S1) for the methylated nucleosomes, but failed to detect any systematic change in DNA geometry or in intermolecular DNA-histone energy related to methylation (Fig. S1B, S1C, S4–S6 in Text S1). The hydrophobic effect should favor orientation of the methyl group out from the solvent but this effect alone is not likely to justify the positional dependent stability changes in Figure 2, as the differential solvation of the methyl groups in the bound and unbound states is only in the order of a fraction of a water molecule (Figure S5 in Text S1). We find however, a reasonable correlation between methylation-induced changes in hydrogen bond and stacking interactions of the bases and the change in nucleosome stability (see Figure S6 in Text S1). This finding suggests that methylation-induced nucleosome destabilization is related to the poorer ability of methylated DNA to fit into the required conformation for DNA in a nucleosome. Changes in the elastic deformation energy between methylated and un-methylated DNA correlate with nucleosomal differential binding free energies To further analyze the idea that methylation-induced nucleosome destabilization is connected to a worse fit of methylated DNA into the required nucleosome-bound conformation, we computed the elastic energy of the nucleosomal DNA using a harmonic deformation method [36,37,44]. This method provides a rough estimate of the energy required to deform a DNA fiber to adopt the super helical conformation in the nucleosome (full details in Suppl. Information Text S1). As shown in Figure 2, there is an evident correlation between the increase that methylation produces in the elastic deformation energy (DDE def.) and the free energy variation (DDG bind.) computed from MD/TI calculations. Clearly, methylation increases the stiffness of the CpG step [31], raising the energy cost required to wrap DNA around the histone octamers. This extra energy cost will be smaller in regions of high positive roll (naked DNA MeCpG steps have a higher roll than CpG steps [31]) than in regions of high negative roll. Thus, simple elastic considerations explain why methylation is better tolerated when the DNA faces the histones through the major groove (where positive roll is required) that when it faces histones through the minor groove (where negative roll is required). Nucleosome methylation can give rise to nucleosome repositioning We have established that methylation affects the wrapping of DNA in nucleosomes, but how does this translate into chromatin structure? As noted above, accumulation of minor groove methylations strongly destabilizes the nucleosome, and could trigger nucleosome unfolding, or notable changes in positioning or phasing of DNA around the histone core. While accumulation of methylations might be well tolerated if placed in favorable positions, accumulation in unfavorable positions would destabilize the nucleosome, which might trigger changes in chromatin structure. Chromatin could in fact react in two different ways in response to significant levels of methylation in unfavorable positions: i) the DNA could either detach from the histone core, leading to nucleosome eviction or nucleosome repositioning, or ii) the DNA could rotate around the histone core, changing its phase to place MeCpG steps in favorable positions. Both effects are anticipated to alter DNA accessibility and impact gene expression regulation. The sub-microsecond time scale of our MD trajectories of methylated DNAs bound to nucleosomes is not large enough to capture these effects, but clear trends are visible in cases of multiple mutations occurring in unfavorable positions, where unmethylated and methylated DNA sequences are out of phase by around 28 degrees (Figure S7 in Text S1). Due to this repositioning, large or small, DNA could move and the nucleosome structure could assume a more compact and distorted conformation, as detected by Lee and Lee [29], or a slightly open conformation as found in Jimenez-Useche et al. [30]. Using the harmonic deformation method, we additionally predicted the change in stability induced by cytosine methylation for millions of different nucleosomal DNA sequences. Consistently with our calculations, we used two extreme scenarios to prepare our DNA sequences (see Fig. 3): i) all positions where the minor grooves contact the histone core are occupied by CpG steps, and ii) all positions where the major grooves contact the histone core are occupied by CpG steps. We then computed the elastic energy required to wrap the DNA around the histone proteins in unmethylated and methylated states, and, as expected, observed that methylation disfavors DNA wrapping (Figure 3A). We have rescaled the elastic energy differences with a factor of 0.23 to match the DDG prediction in figure 2B. In agreement with the rest of our results, our analysis confirms that the effect of methylation is position-dependent. In fact, the overall difference between the two extreme methylation scenarios (all-in-minor vs all-in-major) is larger than 60 kJ/mol, the average difference being around 15 kJ/ mol. We have also computed the elastic energy differences for a million sequences with CpG/MeCpG steps positioned at all possible intermediate locations with respect to the position (figure 3B). The large differences between the extreme cases can induce rotations of DNA around the histone core, shifting its phase to allow the placement of the methylated CpG steps facing the histones through the major groove. It is illustrative to compare the magnitude of CpG methylation penalty with sequence dependent differences. Since there are roughly 1.5e88 possible 147 base pairs long sequence combinations (i.e., (4n+4(n/2))/2, n = 147), it is unfeasible to calculate all the possible sequence effects. However, using our elastic model we can provide a range of values based on a reasonably large number of samples. If we consider all possible nucleosomal sequences in the yeast genome (around 12 Mbp), the energy difference between the best and the worst sequence that could form a nucleosome is 0.7 kj/mol per base (a minimum of 1 kJ/mol and maximum of around 1.7 kJ/mol per base, the first best and the last worst sequences are displayed in Table S3 in Text S1). We repeated the same calculation for one million random sequences and we obtained equivalent results. Placing one CpG step every helical turn gives an average energetic difference between minor groove and major groove methylation of 15 kJ/ mol, which translates into ,0.5 kJ/mol per methyl group, 2 kJ/ mol per base for the largest effects. Considering that not all nucleosome base pair steps are likely to be CpG steps, we can conclude that the balance between the destabilization due to CpG methylation and sequence repositioning will depend on the PLOS Computational Biology | www.ploscompbiol.org 4 November 2013 | Volume 9 | Issue 11 | e1003354 DNA Methylation and Nucleosome Positioning Figure 3. Methylated and non-methylated DNA elastic deformation energies. (A) Distribution of deformation energies for 147 bplong random DNA sequences with CpG steps positioned every 10 base steps (one helical turn) in minor (red and dark red) and major (light and dark blue) grooves respectively. The energy values were rescaled by the slope of a best-fit straight line of figure 2, which is 0.23, to por la lectura a través de la lectura de la prensa. La educación en los medios las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio adquiere pleno derecho y entidad en la sección sexta titulada «competencias sociales y cívi- técnico de los aparatos. cas» que indica que «los alum- nos deberán ser capaces de juz- gar y tendrán espíritu crítico, lo que supone ser educados en los las programaciones oficiales, ya que, a lo largo de un medios y tener conciencia de su lugar y de su influencia estudio de los textos, los documentalistas del CLEMI en la sociedad». han podido señalar más de una centena de referencias a la educación de los medios en el seno de disciplinas 4. Un entorno positivo como el francés, la historia, la geografía, las lenguas, Si nos atenemos a las cifras, el panorama de la las artes plásticas : trabajos sobre las portadas de educación en medios es muy positivo. Una gran ope- prensa, reflexiones sobre temas mediáticos, análisis de ración de visibilidad como la «Semana de la prensa y publicidad, análisis de imágenes desde todos los ángu- de los medios en la escuela», coordinada por el CLE- los, reflexión sobre las noticias en los países europeos, MI, confirma año tras año, después de 17 convocato- información y opinión rias, el atractivo que ejerce sobre los profesores y los Esta presencia se constata desde la escuela mater- alumnos. Concebida como una gran operación de nal (2 a 6 años) donde, por ejemplo, se le pregunta a complementariedad entre la escuela y los profesiona- los niños más pequeños si saben diferenciar entre un les de los medios, alrededor del aprendizaje ciudada- periódico, un libro, un catálogo, a través de activida- no de la comunicación mediática, este evento moviliza des sensoriales, si saben para qué sirve un cartel, un durante toda una semana un porcentaje elevado de periódico, un cuaderno, un ordenador si son capa- centros escolares que representan un potencial de 4,3 ces de reconocer y distinguir imágenes de origen y de millones de alumnos (cifras de 2006). Basada en el naturaleza distintas. Podríamos continuar con más voluntariado, la semana permite desarrollar activida- ejemplos en todos los niveles de enseñanza y práctica- des más o menos ambiciosas centradas en la introduc- Páginas 43-48 ción de los medios en la vida de la escuela a través de la instalación de kioscos, organización de debates con profesionales y la confección por parte de los alumnos de documentos difundidos en los medios profesionales. Es la ocasión de dar un empujón a la educación en medios y de disfrutarlos. Los medios –un millar en 2006– se asocian de maneras diversas ofreciendo ejemplares de periódicos, acceso a noticias o a imágenes, proponiendo encuentros, permitiendo intervenir a los jóvenes en sus ondas o en sus columnas Esta operación da luz al trabajo de la educación en medios y moviliza a los diferentes participantes en el proyecto. 5. La formación de los docentes La formación es uno de los pilares principales de la educación en los medios. Su función es indispensable ya que no se trata de una disciplina, sino de una enseñanza que se hace sobre la base del voluntariado y del compromiso personal. Se trata de convencer, de mostrar, de interactuar. En primer lugar es necesario incluirla en la formación continua de los docentes, cuyo volumen se ha incrementado desde 1981 con la aparición de una verdadera política de formación continua de personal. Es difícil dar una imagen completa del volumen y del público, pero si nos atenemos a las cifras del CLEMI, hay más de 24.000 profesores que han asistido y se han involucrado durante 2004-05. 5.1. La formación continua En la mayoría de los casos, los profesores reciben su formación en contextos cercanos a su centro de trabajo, o incluso en este mismo. Después de una política centrada en la oferta que hacían los formadores, se valora más positivamente la demanda por parte del profesorado, ya que sólo así será verdaderamente fructífera. Los cursos de formación se repartieron en varias categorías: desde los formatos más tradicionales (cursos, debates, animaciones), hasta actividades de asesoramiento y de acompañamiento, y por supuesto los coloquios que permiten un trabajo en profundidad ya que van acompañados de expertos investigadores y profesionales. Citemos, por ejemplo en 2005, los coloquios del CLEMI-Toulouse sobre el cine documental o el del CLEMI-Dijon sobre «Políticos y medios: ¿connivencia?». Estos coloquios, que forman parte de un trabajo pedagógico regular, reagrupan a los diferentes participantes regionales y nacionales alrededor de grandes temas de la educación en medios y permiten generar nuevos conocimientos de aproximación y una profundización. Páginas 43-48 Hay otro tipo de formación original que se viene desarrollando desde hace menos tiempo, a través de cursos profesionales, como por ejemplo, en el Festival Internacional de Foto-periodismo «Visa para la imagen», en Perpignan. La formación se consolida en el curso, da acceso a las exposiciones, a las conferencias de profesionales y a los grandes debates, pero añade además propuestas pedagógicas y reflexiones didácticas destinadas a los docentes. Estas nuevas modalidades de formación son también consecuencia del agotamiento de la formación tradicional en las regiones. Los contenidos más frecuentes en formación continua conciernen tanto a los temas más clásicos como a los cambios que se están llevando a cabo en las prácticas mediáticas. Así encontramos distintas tendencias para 2004-05: La imagen desde el ángulo de la producción de imágenes animadas, el análisis de la imagen de la información o las imágenes del J.T. La prensa escrita y el periódico escolar. Internet y la información en línea. Medios y educación de los medios. 5.2 La formación inicial La formación inicial está aun en un grado muy ini- cial. El hecho de que la educación en medios no sea una disciplina impide su presencia en los IUFM (Institutos Universitarios de Formación de Maestros) que dan una prioridad absoluta a la didáctica de las disciplinas. En 2003, alrededor de 1.400 cursillistas sobre un total de 30.000 participaron en un momento u otro de un módulo de educación en medios. Estos módulos se ofrecen en función del interés que ese formador encuentra puntualmente y forman parte a menudo de varias disciplinas: documentación, letras, historia-geografía Estamos aún lejos de una política concertada en este dominio. La optativa «Cine-audiovisual» ha entrado desde hace muy poco tiempo en algunos IUFM destinada a obtener un certificado de enseñanza de la opción audiovisual y cine. Internet tiene cabida también en los cursos de formación inicial, recientemente con la aparición de un certificado informático y de Internet para los docentes, dirigido más a constatar competencias personales que a valorar una aptitud para enseñarlos. 6. ¿Y el futuro? El problema del futuro se plantea una vez más por la irrupción de nuevas técnicas y nuevos soportes. La difusión acelerada de lo digital replantea hoy muchas cuestiones relativas a prácticas mediáticas. Muchos Comunicar, 28, 2007 47 Comunicar, 28, 2007 Enrique Martínez-Salanova '2007 para Comunicar 48 trabajos que llevan el rótulo de la educación en medios solicitan una revisión ya que los conceptos cambian. La metodología elaborada en el marco de la educación en medios parece incluso permitir la inclinación de la sociedad de la información hacia una sociedad del conocimiento, como defiende la UNESCO. En Francia, se necesitaría unir las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio técnico de los aparatos. Los avances recientes en el reconocimiento de estos contenidos y las competencias que supondrían podrían permitirlo. Referencias CLEMI/ACADEMIE DE BORDEAUX (Ed.) (2003): Parcours médias au collège: approches disciplinaires et transdisciplinaires. Aquitaine, Sceren-CRDP. GONNET, J. (2001): Education aux médias. Les controverses fécondes. Paris, Hachette Education/CNDP. SAVINO, J.; MARMIESSE, C. et BENSA, F. (2005): L’éducation aux médias de la maternelle au lycée. Direction de l’Enseignement Scolaire. Paris, Ministère de l’Education Nationale, Sceren/CNDP, Témoigner. BEVORT, E. et FREMONT, P. (2001): Médias, violence et education. Paris, CNDP, Actes et rapports pour l’éducation. – www.clemi.org: fiches pédagogiques, rapports et liens avec les pages régionales/académiques. – www.ac-nancy-metz.fr/cinemav/quai.html: Le site «Quai des images» est dédié à l’enseignement du cinéma et de l’audiovisuel. – www.france5.fr/education: la rubrique «Côté profs» a une entrée «education aux médias». – www.educaunet.org: Programme européen d’éducation aux risques liés à Internet. dResedfeleexliobnuetsacón Páginas 43-48
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