Biofield Treated

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Orchids are used worldwide for indoor decoration, vanilla production, and beverage preparation. They are also reported for their therapeutic efficacy in brain-related problems. The in vitro micropropagation technique was used for their propagation using the orchid maintenance/replate (OMR) medium.

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Journal of Plant Sciences 2015 36: 285-293 Published online November 2015 http://www.sciencepublishinggroup.com/j/jps doi: 10.11648/j.jps.20150306.11 ISSN: 2331-0723 Print ISSN: 2331-0731 Online Physicochemical Characterization of Biofield Treated Orchid Maintenance/Replate Medium Mahendra Kumar Trivedi 1 Alice Branton 1 Dahryn Trivedi 1 Gopal Nayak 1 Ragini Singh 2 Snehasis Jana 2 1 Trivedi Global Inc. Henderson NV USA 2 Trivedi Science Research Laboratory Pvt. Ltd. Bhopal Madhya Pradesh India Email address: publicationtrivedisrl.com S. Jana To cite this article: Mahendra Kumar Trivedi Alice Branton Dahryn Trivedi Gopal Nayak Ragini Singh Snehasis Jana. Physicochemical Characterization of Biofield Treated Orchid Maintenance/Replate Medium. Journal of Plant Sciences. Vol. 3 No. 6 2015 pp. 285-293. doi: 10.11648/j.jps.20150306.11 Abstract: Orchids are used worldwide for indoor decoration vanilla production and beverage preparation. They are also reported for their therapeutic efficacy in brain-related problems. The in vitro micropropagation technique was used for their propagation using the orchid maintenance/replate OMR medium. The current study was based on analysing the effect of biofield energy treatment on the physicochemical properties of OMR medium. A part of the sample was treated with Mr. Trivedi’s biofield energy various physicochemical properties were analyzed and compared with the untreated control part. The X-ray diffraction analysis revealed the decrease in crystallite size of treated sample 132.80 nm as compared to the control 147.55 nm. The particle size analysis revealed 20.78 increase in average particle size and 39.29 increase in d 99 size below which 99 particles are present of the treated OMR medium as compared to the control. Moreover the surface area of the treated sample was reduced by 3.9 supporting the data of particle size analysis. The thermal analysis studies revealed an increase in the thermal stability of the treated OMR medium as compared to the control. The analysis was done by using differential scanning calorimetry that showed increase in melting point 1.23 and latent heat of fusion 135.7 and thermogravimetric analysis that reported increase in onset temperature and maximum thermal degradation temperature of the treated sample as compared to the control. Besides the CHNSO analysis revealed the increase in percentage of nitrogen 22.22 as well as the presence of sulphur in the treated sample. The Fourier transform infrared and UV-visible spectroscopy also showed the differences in the spectra of the treated sample as compared to the control OMR medium. Hence the overall data revealed the impact of biofield energy treatment on the physicochemical properties of the treated sample that might be used in better way in the in vitro culture techniques as compared to the control sample. Keywords: Orchid Maintenance/Replate Medium Biofield Energy Treatment In vitro Micropropagation Complementary and Alternative Medicines 1. Introduction Orchids belonging to family Orchidaceae are widely used due to their medicinal properties in several countries around the world 1. Several kind of research had shown their therapeutic efficacy in case of hysteria nervous irritability and other brain related dysfunctions. Besides several species are reported as a febrifuge clearing tapeworms treating skin diseases etc. 2 3. It was reported that the biological activity was due to the presence of alkaloids such as strychnine morphine nicotine reserpine etc. 4. Moreover the orchid family is also important for its horticulture uses. It is used for commercial production of vanilla 5. In Turkey it is used in the preparation of a traditional beverage called as salep 3. It has great importance as cut flowers and indoor decoration. The general method of propagation of orchid is asexual but it can produce only 2-4 plants per year as it is very slow process 6 7. Hence in vitro micropropagation technique is frequently used that produces plantlets using tissue culture techniques 8. Micropropagation is the process used to replicate the plant using plant seed or tissue in the laboratory under the sterile conditions 9. The most important factor in successful tissue culture of plant cells is

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286 Mahendra Kumar Trivedi et al.: Physicochemical Characterization of Biofield Treated Orchid Maintenance/Replate Medium the composition of the medium. It provides the essential nutrients for the survival of plant cells or tissues and the optimum conditions such as pH osmotic pressure etc. 10. Orchid maintenance/replate OMR medium is such type of medium that consists of accurately defined organic and inorganic chemical additives in the form of macro- and micronutrients 11. The constituents of the medium are shown in Table 1. It differs from original orchid replate medium as it contains banana powder to promote rooting and growth and agar as a gelling agent that provides physical support 12. Despite several advantages it suffers the problem of hygroscopicity due to which it needs to be protected from atmospheric moisture and proper storage conditions 2-8°C 13. Hence some alternative is needed that can improve its properties thereby its use as orchid micropropagation medium. Table 1. Components of the orchid maintenance/replate medium. S. No. Ingredient milligram/litre 1 Potassium nitrate 950.00 2 Ammonium nitrate 825.00 3 Calcium chloride.2H 2O 220.00 4 Magnesium sulphate 90.34 5 Potassium phosphate monobasic 85.00 6 Manganese sulphate.H 2O 8.45 7 Boric acid 3.10 8 Potassium iodide 0.42 9 Molybdic acid sodium salt.2H 2O 0.13 10 Zinc sulphate.7H 2O 5.30 11 Copper sulphate.5H 2O 0.0125 12 Cobalt chloride.6H 2O 0.0125 13 Ferrous sulphate.7H 2O 27.80 14 EDTA disodium salt.2H 2O 37.30 15 myo - Inositol 100.00 16 Thiamine hydrochloride 10.00 17 Pyridoxine hydrochloride 1.00 18 Nicotinic acid Free acid 1.00 19 Peptone 2000.00 20 Banana powder 30000.00 21 Sucrose 20000.00 22 MES 1000.00 23 Agar 7000.00 24 Activated charcoal 2000.00 MES: 2-n-morpholinoethanesulfonic acid EDTA: Ethylenediamine tetraacetic acid The biofield energy treatment is reported to affect the health of human beings via interacting with their biofield 14. The National Centre for Complementary and Alternative Medicine NCCAM which is the part of National Institute of Health NIH also include the energy medicines as complementary and alternative medicines CAM 15. It is a putative form of energy that is produced by own emissions of the body and surrounds the body of all living organisms. However the frequency of this energy depends on the physiological and mental state of the person. The living systems are continuously exchanging this energy with their surroundings to maintain themselves 16 17. The non-living things also possess the biofield energy as everything in the universe made up of same constituents however they are not able to change their energy aura by more than 2 18. Thus the human has the ability to harness the energy from the environment and can transmit it to any living or non-living object. Mr. Trivedi is also known to possess unique biofield energy and the treatment is called as The Trivedi Effect ® . It is known for its impact on various living organisms and non-living materials including microorganisms 19 pharmaceutical compounds 20 and yield growth and anatomical characteristics of plants 21 22. Hence based on the importance of OMR medium and the outcomes of the biofield energy treatment the study was designed to analyse the impact of Mr. Trivedi’s biofield energy treatment on various physicochemical properties of the OMR medium. 2. Materials and Methods Orchid maintenance/replate OMR Medium was procured from HiMedia Laboratories India. The sample was divided into two parts and coded as control and treated. Mr. Trivedi’s biofield energy treatment was provided to the treated part while no treatment was given to the control part. For treatment the treated part was handed over to Mr. Trivedi in sealed pack under standard laboratory conditions. Mr. Trivedi provided the treatment to the treated part through his unique energy transmission process without touching the sample. The control and biofield treated samples were further characterised using various analytical techniques. 2.1. X-ray Diffraction XRD Study The Phillips Holland PW 1710 X-ray diffractometer system was used to obtain the X-ray powder diffractogram of control and treated samples. The X-ray generator was equipped with a copper anode with nickel filter operating at 35kV and 20mA. The XRD system used 1.54056Å wavelength of radiation. The data were collected from the 2θ range of 10°-99.99° and a counting time of 0.5 seconds per step along with a step size of 0.02°. The crystallite size G was calculated from the Scherrer equation: G kλ/bCosθ Where k is constant 0.94 λ is the X-ray wavelength 0.154 nm b in radians is the full-width at half of the peak and θ is the corresponding Bragg’s angle. Moreover the percent change in crystallite size was calculated using the following equation: Percent change in crystallite size G t -G c /G c ×100 Here G c and G t denotes the crystallite size of control and treated powder samples respectively. 2.2. Particle Size Analysis For particle size analysis laser particle size analyzer SYMPATEC HELOS-BF was used having a detection range of 0⋅1-875 µm. Two parameters of particle sizes viz. d 50 and d 99 size below which 50 and 99 particles are

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Journal of Plant Sciences 2015 36: 285-293 287 present respectively were calculated. The percent change in average particle size d 50 was calculated using following equation: change in particle sized d − d d ×100 Where d 50 Control and d 50 Treated represents the average particle size of control and treated samples respectively. Similarly the percent change in particle size d 99 was calculated. 2.3. Surface Area Analysis The surface area was measured by the Brunauer–Emmett– Teller BET surface area analyser Smart SORB 90. The percent change in surface area was calculated using following equation: change in surface area S −S S ×100 Where S Control and S Treated are the surface area of control and treated samples respectively. 2.4. Thermal Analysis The thermal stability profile of OMR medium was analyzed using DSC and TGA/DTG studies. The impact of biofield treatment was analyzed by comparing the results of treated sample with that of the control sample. 2.4.1. Differential Scanning Calorimetry DSC Study The DSC analysis of control and treated samples was carried out using Perkin Elmer/Pyris-1. The samples were heated at a rate of 10°C/min under air atmosphere 5 mL/min. The thermograms were collected over the temperature range of 50°C to 300°C. 2.4.2. Thermogravimetric Analysis/Derivative Thermogravimetry TGA/DTG The effect of temperature on the stability of the control and treated sample of OMR medium was analyzed using Mettler Toledo simultaneous thermogravimetric analyser TGA/DTG. The heating temperature was selected from room temperature to 350ºC with a heating rate of 5ºC/min under air atmosphere. 2.5. CHNSO Analysis The control and treated samples of OMR medium were analyzed using CHNSO analyzer using Model Flash EA 1112 series Thermo Finnigan Italy. 2.6. Fourier Transform-Infrared FT-IR Spectroscopic Characterization For FT-IR characterization the samples were crushed mixed with spectroscopic grade KBr and pressed into pellets with a hydraulic press. The FT-IR spectra were recorded on Shimadzu’s Fourier transform infrared spectrometer Japan in the frequency range 4000-350 cm -1 . The FT-IR spectral analysis was used to determine the effect of biofield energy on the strength of bonds and stability of compounds present in OMR medium. 2.7. UV-Vis Spectroscopic Analysis The UV-Vis spectral analysis was measured using Shimadzu UV-2400 PC series spectrophotometer. The spectrum was recorded using 1 cm quartz cell that has a slit width of 2.0 nm. 3. Results and Discussion 3.1. X-Ray Diffraction XRD The X-ray powder diffractograms of control and treated samples of OMR medium showed a series of sharp peaks in the regions of 10º2θ40º. In the control sample the peaks were observed at 2θ equal to 11.67° 16.72° 18.72° 18.88° 19.64° 21.49° and 25.23°. However the treated sample showed the peaks at 2θ equal to 11.61° 13.05° 16.67° 17.73° 18.74° 22.04° 24.64° and 25.17°. In addition the most intense peak in control sample was observed at 2θ equal to 25.23° however in treated sample it was observed at 22.04°. It indicated that the relative intensities of XRD peaks were altered in the treated OMR medium as compared to the control sample. Besides the crystallite size of the control sample was found as 147.55 nm whereas in the treated sample it was found as 132.80 nm. It suggested that crystallite size of the treated sample was significantly decreased by 10 as compared to the control. The changes in the relative intensities of peaks revealed the presence of microstrain may be due to the biofield treatment. It may result in change in dislocation densities and atomic displacements that might be the reason for decreased crystallite size 23 24. 3.2. Particle Size Analysis The particle size of control and treated samples of OMR medium are presented in Fig. 1. It showed that the d 50 and d 99 were 26.03 and 236.92 µm respectively in the control sample. However in treated sample the d 50 and d 99 were found as 31.44 and 330.01 µm respectively. It revealed that d 50 was increased by 20.78 and d 99 was increased by 39.29 in the treated sample as compared to the control. The temperature has a significant effect on the particle size of the sample 25. Hence it is presumed that the biofield treatment may provide some energy to the sample that resulted in increased particle size as compared to the control sample. Moreover the particle size was directly related to the viscosity and gelling property of the compound 26. Hence the treated OMR medium sample with increased particle size might improve the gelling property of media as the large particles has less tendency to broken down and has stronger water holding capacity as compared with the small particles.

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288 Mahendra Kumar Trivedi et al.: Physicochemical Characterization of Biofield Treated Orchid Maintenance/Replate Medium Fig. 1. Particle size analysis of control and treated samples of OMR medium. 3.3. Surface Area Analysis The surface area of control and treated samples of OMR medium was investigated using BET method. The control sample showed a surface area of 2.327 m 2 /g however the treated sample of OMR medium showed a surface area of 2.236 m 2 /g. It showed that the surface area was decreased by 3.91 in the treated OMR medium sample as compared to the control. The decrease in surface area of treated OMR medium sample may be due to the increase in the particle size as evident from the particle size analysis. Besides the OMR medium has the problem of hygroscopicity 13 and the surface area was directly related to the hygroscopic water content of the sample 27. Hence it is assumed that the treated OMR medium sample with decreased surface area might reduce the problem of hygroscopicity as compared with the control sample. 3.4. Thermal Analysis 3.4.1. DSC Analysis Fig. 2. DSC analysis of control and treated samples of OMR medium. The DSC thermograms of control and treated samples of OMR medium are presented in Fig. 2. The thermogram of control sample showed an endothermic peak at 180.36°C due to the melting of the sample. In this process the amount of heat absorbed latent heat of fusion ∆H was recorded as 23.00 J/g. The similar endothermic peak was observed in

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Journal of Plant Sciences 2015 36: 285-293 289 treated sample at 182.58°C and ∆H was recorded as 54.20 J/g. The result of DSC analysis revealed slight alteration 1.23 increase in the melting temperature along with 135.7 increase in ∆H. The particle size can influence the melting temperature and ∆H of the corresponding sample as it is directly related to the melting properties 28 29. Hence it might be a reason for the increase in melting temperature and ∆H of the OMR medium as the particle size was found increased after the biofield treatment. 3.4.2. TGA/DTG Analysis The TGA/DTG studies analyse the pattern of thermal decomposition of the sample during heating. The TGA/DTG thermograms of the control and treated samples of OMR medium are presented in Fig. 3. The thermogram of control sample showed the degradation of the sample in three steps. Moreover the first step degradation of the sample was started at 175.72°C and ended at 203.38°C. Besides the treated sample showed two-step degradation where the first step commenced at 187.61°C and completed at 237.96°C. It indicated that the onset temperature of degradation was increased in the treated sample as compared to the control. Besides DTG thermogram data showed that T max was observed at 189.19°C in the control sample while 210.41°C in the treated OMR medium. It indicated that T max was increased by approximately 21°C in the treated sample as compared to the control. Furthermore the increase in onset temperature of decomposition and T max in the treated sample of OMR medium with respect to the control sample may be correlated with the increased thermal stability. The particle size has a significant impact on the onset and peak temperature and they were found directly proportional to each other 29. Hence the increase in particle size of OMR medium after treatment might be a reason for the increase in thermal stability. Besides it is well known that the OMR medium faces high-temperature treatment e.g. autoclaving before used as culture media where it may suffer from the problem of thermal degradation. Hence the treated sample with increased thermal stability might help in increasing the efficacy and shelf-life of the treated sample as compared to the control. Fig. 3. TGA/DTG analysis of control and treated samples of OMR medium. 3.5. CHNSO Analysis The CHNSO analysis was used to measure the percentage of elements present in the given sample. The result of CHNSO analysis of control and treated samples are presented in Table 2. The data revealed that the percentage of nitrogen was significantly increased by 22.22 whereas the

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290 Mahendra Kumar Trivedi et al.: Physicochemical Characterization of Biofield Treated Orchid Maintenance/Replate Medium percentage of carbon hydrogen and oxygen was slightly decreased as 3.62 8.51 and 1.92 respectively in the treated sample as compared to the control. Besides the treated sample showed the presence of sulphur that was not detected in the control sample. It is well known that nitrogen is the main component of media that is provided by ammonium nitrate potassium nitrate and peptone. The increased percentage of nitrogen in the treated sample may help to improve the growth of orchid culture as compared with the control. Table 2. CHNSO data of orchid maintenance / replate medium. Element Control Treated Percent change Nitrogen 0.63 0.77 22.22 Carbon 43.90 42.31 -3.62 Hydrogen 6.93 6.34 -8.51 Oxygen 31.18 30.58 -1.92 Sulphur ND 0.27 ND: not detected 3.6. FT-IR Spectroscopic Analysis Fig. 4. FT-IR spectra of control and treated samples of OMR medium. The FT-IR spectra of OMR medium control and treated samples are shown in Fig. 4. The sample contains several ingredients such as ammonium nitrate disodium EDTA ferrous sulphate potassium nitrate nicotinic acid sucrose inositol thiamine hydrochloride and pyridoxine hydrochloride etc. Hence the major vibration peaks were observed Table 3 related to the functional groups present in these ingredients. The peak at 3336 cm -1 in the control sample was assigned to N-H stretching of ammonium nitrate and O-H stretching carboxylic acid due to nicotinic acid and disodium EDTA 30 31 however the broadness of peak suggests the hydrogen bonding within the compound. Besides in the treated sample it was shifted to a lower frequency at 3323 cm -1 . Further the C-H stretching peaks of disodium EDTA appeared at 2983 and 2896 cm -1 in the control sample whereas in the treated sample the peaks

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Journal of Plant Sciences 2015 36: 285-293 291 appeared at 2941 and 2898 cm -1 . The peak due to pyridine ring of nicotinic acid and pyridoxine HCl was observed at 2815 cm -1 in the control while 2833 cm -1 in the treated sample. Similarly the peak at 1753 cm -1 in the control sample was assigned to CO stretching of lactone ring present in sucrose however it was observed at 1735 cm -1 in the treated sample 30. The peak at 1595 cm -1 in the control sample appeared as doublet and it was assigned to ring stretching of the pyridine ring of nicotinic acid and pyridoxine HCl 32. The peak may also merge with the peak due to S-O bond of CuSO 4 and P-O bond of KPO 4 31. Besides in the treated sample the corresponding peak was observed as a singlet at 1608 cm -1 . Furthermore the peak at 1380 cm -1 in the control that was shifted to 1417 cm -1 in the treated sample was assigned to N-O symmetric stretching of KNO 3 and pyrimidine ring of thiamine HCl 31 32. The peaks at 1234 cm -1 in the control sample and 1236 cm -1 in the treated sample was assigned to thiazole ring breathing of the thiamine HCl 33. Moreover the peak at 1146 and 1130 cm -1 in the control and treated samples respectively was assigned to S-O bond in FeSO 4 and ZnSO 4 and pyrimidine ring of thiamine HCl. The peak due to C-O stretching of alcohol group in pyridoxine HCl was observed at 1047 cm -1 in the control and 1058 cm -1 in the treated sample. The ring breathing mode of inositol was observed at 1001 cm -1 in the control and 999 cm -1 in the treated sample. Further the peak at 858 cm -1 in both control and treated sample was assigned to B-O bond of boric acid and C-H out of plane bending of thiazole ring in thiamine HCl. The IR peaks of control sample were well matched with the reported literature. The FT-IR spectra of the treated sample showed different IR frequencies of respective functional groups as compared to the control. It suggested the impact of biofield energy treatment on the bond strength and dipole moment of the compounds present in the OMR medium. However further studies are needed to analyse the effect of this treatment on the specific compounds and their functions in OMR medium. Table 3. Vibration modes observed in orchid maintenance/replate medium. S. No. Functional group Compound Wavenumber cm -1 Control Treated 1 O-H stretching N-H stretching Nicotinic acid Disodium EDTA Ammonium nitrate 3336 3323 2 C-H stretching Disodium EDTA Thiamine HCl 2983 2896 2941 2898 3 C-H stretching Agar 2815 2833 4 CO stretching lactone Sucrose 1753 1735 5 Ring stretching pyridine Nicotinic acid Pyridoxine HCl 1595 1608 6 N-O stretching Ring stretching pyrimidine KNO 3 Thiamine HCl 1380 1417 7 Ring breathing thiazole SO stretching Thiamine HCl MES 1234 1236 8 Pyrimidine ring stretching S-O bond Thiamine HCl FeSO 4 ZnSO 4 1146 1130 9 C-O stretching C- OH Pyridoxine HCl 1047 1058 10 Ring breathing carbon ring Inositol 1001 999 11 B-O stretching Boric acid 858 858 12 Ring deformation cycloalkane Inositol 561 551 3.7. UV-Vis Spectroscopic Analysis The UV spectra of OMR medium control and treated samples are shown in Fig. 5. The UV spectrum of control sample showed absorption peaks at λ max equal to 212 and 257 nm. However the biofield treated sample showed absorption peaks at λ max equal to 212 and 275 nm. The peak at λ max 257 nm in the control sample was shifted to higher wavelength i.e. 275 nm in the treated sample. It is hypothesized that biofield energy treatment might affect the HOMO→LUMO transition within the compounds of OMR medium due to which the peak at λ max 257 nm was shifted to 275 nm in the treated sample. Fig. 5. UV-Vis spectra of control and treated samples of OMR medium.

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292 Mahendra Kumar Trivedi et al.: Physicochemical Characterization of Biofield Treated Orchid Maintenance/Replate Medium 4. Conclusions The XRD study showed 10 decrease in the crystallite size of treated sample along with alteration in the relative intensities of the peaks. It may occur due to the presence of microstrains that might be generated after biofield energy treatment. Moreover the average particle size and d 99 were increased in treated sample by 20.78 and 39.29 respectively as compared to the control. The surface area data supported the results of particle size analysis and revealed that the surface area was decreased by 4 in the treated sample. The increased particle size and reduced surface area might improve the gelling properties and reduce the problem of hygroscopicity of the treated sample. Besides the melting temperature and ∆H was found increased in the treated sample as compared to the control. The TGA results also revealed that the onset temperature of degradation and maximum degradation temperature was increased in the treated sample. The increased thermal stability may help in increasing the efficacy and shelf-life of the treated sample as compared to the control. Furthermore the CHNSO analysis revealed increased percent of nitrogen along with the presence of sulphur in the treated sample as compared to the control. The FT-IR and UV-vis spectra of the treated sample also revealed the changes as compared to the control. The overall study revealed the impact of biofield treatment on the physical thermal and spectroscopic properties of the OMR medium that could make it more useful as compared to the control. Acknowledgements Authors greatly acknowledge the support of Trivedi Science Trivedi Master Wellness and Trivedi Testimonials in this research work. The authors would also like to acknowledge the whole team from the Sophisticated Analytical Instrument Facility SAIF Nagpur and MGV Pharmacy College Nashik for providing the instrumental facility. References 1 Atwood JT 1986 The size of the orchidaceae and systematic position of epiphytic orchids. Selbyana 9: 171-186. 2 Jalal JS Kumar P Tewari L Pangtey YPS. Orchids: Uses in traditional medicine in India. National seminar on medicinal plants of Himalaya: Potential and prospect. Regional Research Institute of Himalayan Flora Tarikhet India. 3 Bulpitt CJ 2005 The uses and misuses of orchids in medicine. QJM 98: 625-631. 4 Bulpitt CJ Li Y Bulpitt PF Wang J 2007 The use of orchids in chinese medicine. J R Soc Med 100: 558-563. 5 Sforza S 2013 Food authentication using bioorganic molecules. DEStech Publications Inc. USA. 6 Khatun H Khatun MM Biswas MS Kabir MR Al-Amin M 2010 In-vitro growth and development of Dendrobium hybrid orchid. 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