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Introduction

Sphagnum is one of the of import species in the Earth ; the genus peat moss has approximately 100 to 300 species. Sphagnum genus is the major component of ombrotrophic bogs contains big sum of life and dead biomass ( Van Breemen, 1995, Jim Provan and Pamela J. Wilson ) . Peat lands screens about one tierce of the planetary C ( C ) ( Gorham 1991 ) . Sphagnum is the major species in peat lands contains big sum of C in locked signifier. Sphagnum is comprise of secondary compounds such as polyphenols and uronic acid, due to this curious characters sphagnum is immune to decomposition of organic affair due to microbes ( painter 1991, Verhoevn and Liefveld, 1997, Freeman et al 2001 ) and the decomposition rate of peat moss is really less compared to other workss. The C is stored in peat moss in the signifier of polyphenols. It has a history of use since from epochs to continue the dead organic structures, cheese, etc. due to the curious characters of the peat moss and it is widely used medicinally. They are the secondary metabolites produced in the phenylpropanoid tract in presence of the enzymes phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase as cardinal enzymes. Phenylpropanoids are the largest secondary metabolites produced by the workss because of biotic or abiotic emphasiss, UV radiation, temperature and exposure to other environmental conditions ( L.G. Korkina, 2007 ) . All species of peat moss examined are contains high concentrations of the Trans-sphagnum acid, it is synthesized from phenylalanine via the phenylpropanoid tract ( Rasmussen et al 1995 ) . Assorted cistrons are responsible for the alterations in peat moss. PAL and C4H cistrons are major cistrons responsible for production of phenylalanine ammonium hydroxide lyase and cinnamic acid 4-hydroxylase. FAH1 and F3H are responsible for ferulate-5-hydroxylase and flavanone-3-hydroxylase. Atmospheric N ( N ) besides has important consequence on the polyphenols, that is increasing degrees of N may be leads to depletion of polyphenols and increases the decomposition rate of peat moss. However the C locked in the peat moss releases in to the ambiance may take to harmful effects and drastically changes the planetary temperatures. So it is necessary to command this. In order to command the decomposition of peat moss or to suppress the synthesis of phenolic compounds by bring forthing the transgenic workss of peat moss it necessary to cognize about the polyphenols and their tracts, cistrons commanding the phenylpropanoid tract and their look rates. There are less surveies available about the peat moss so it is necessary take some other workss which already studied, for illustration Arabidopsis as theoretical account species. And besides it is of import to cognize the cistrons coding for the enzyme which is responsible for the synthesis of trans-sphagnum acid by utilizing the methods which are suited for the cistron cloning and analysis.

Global heating

The increasing temperatures on the surface of Earth is said to be planetary heating. It is due to the nursery gases ( H2O vapour, C dioxide ( CO2 ) , methane ( CH4 ) , ozone ( O3 ) etc. ) , human activities are major among this, C compounds ( E.g. : Carbon dioxide, CH4. ) are of import nursery gases. Decomposition of the works waste is one of the cardinal beginnings for the C. Peat lands are big pools of C. The storage of the C depends in relation to production and decomposition ( Andy Siegenthaler et al. , 2010 ) . The peat moss genus and its species are the major beginning for the sequestering C and its merchandises. The fallowing graph ( figure 1 ) show that the degrees of the C dioxide is increasing tremendously, and it stressing that the increasing concentration of CO2 at the same time altering the temperature degrees.

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Figure 1: Global C dioxide ( CO2 ) and temperature degrees Vs Time.

Carbon storage in peat lands

In the boreal part most of the peat lands are available and it covers about 10 % of universe vegetive land and contains about 30 % of locked C ( Dixon et al, 1994 ; Vasander and kettunen 2006 ) in the universe entire dirt ( Post et al. , 1982 ) . The accrued Cs from the last glacial upper limit and peat lands are high in the boreal part of the Northern Hemispheres holding the atmospheric CO2 is 600ppm ( Adams et al. , 1990 ) . In the peat mosses genus peat moss contains big sum of life and dead biomass because Sphagnum is a major wetland works in the northern peat lands, about one per centum of the Earth covered with sphagnum moss or peat moss peat moss and one tierce of the planetary C stored in the peat lands. Sphagnum mosses hold the C compounds as in locked signifier. It contains the peculiar polyphenols are derived from the cinnamic acid derived function called trans-sphagnum acid and it produce assorted secondary metabolites. The compounds chiefly synthesized due to the external emphasiss. The peat lands composed of rich in sourness degree and mineral hapless conditions ( Etherington, 1983 ) , due to this the decomposition of the peat moss is non possible and they are stable for old ages. Because of this ground the C and its compounds still remains in the peat mosses. The peat moss forms a mat like signifier which are of course found in bogs and some of the drifting mats blocks the drainage and maintains the bog temperatures ( Crum, 1976 ; Niering, 1966 ) and due to their absorptive nature they besides forms the sponge like mass, in spring it act as dielectric for ice to forestall runing. As we know the temperatures on the Earth are increasing vastly, may be due to human mistakes or environmental factors and increasing degrees it may take to increases the decomposition of peat mosses. If the locked C and its compounds in peat moss decompose and release into the ambiance, it leads instability in the C ecosystem and may perchance duplicate the present C degrees and increases the planetary temperatures and radiation highly, which may demo highly unsafe effects on the all types of beings populating on the Earth.

Sphagnum life rhythm

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Figure 2: The life rhythm procedure of the genus peat moss

Sphagnum is the genus in most of the mosses ; it belongs to the household Sphagnaceae and a division of nonvascular plant. The life rhythm of peat moss is rather different from other works species. The mosses non bear the flowers or seeds. They germinate from the spores and farther develop as a fruit like construction called capsules. Sphagnum consists of two different coevalss of the life rhythm they are gametophyte and sporophyte ( figure-1 ) . In the gametophyte the works bears leaves, synthesizes the chlorophyll and it is monoploid, it can be able to bring forth sexually, where peat moss bring forth egg and sperm on the subdivisions of the same or different workss. The sporophyte is a diploid coevals, which consists of little black capsules on the vertex attached to gametophyte and leaves. The spores are enclosed in the capsules and ejects when they are dried. The sporophytes generated from the fertilized ovum by sexual reproduction. The haploids produced from the diploid by the procedure of miosis. The sporophyte normally spread by distributing the fragments and budding in new workss ( figure-2 ) , because most of the species do non reproduce sexually. In some species the fragments persist to dry for long clip and still hold the nature to turn when H2O is available, in other species once fragments becomes dry about a hebdomad, they can non turn further ( e.g. S.capillifolium ) ( McQueen 1990 ) . The spores are spread by air. Some workss spread their coevals and growing from the subdivisions.

1 2 3

Figure 3: The spreading of the peat moss from the buds by scattering the spores and from subdivisions. ( 1- peat moss works bears the buds, 2-spreading the spores from the buds and 3-spreding of the peat moss from subdivisions )

Sphagnum nature and distribution

Sphagnum mosses are available in two types, peat moss moss and peat moss peat moss and they are different. Sphagnum moss is the populating signifier of the peat moss genus and peat moss peat moss is the dead mass which accumulates the lower degrees of the peat moss. The dead Sphagnum has the nature of keeping the H2O. It is the major wetland works, it normally grows on the wet countries or where the wet content is more. It has a history more than 10,000 twelvemonth, in olden yearss the acidic peat moss bogs are used to continue the mammalian organic structures for ages. These organic structures are said to be peat organic structures for illustration Tollund Man, Haraldskaer Women, Lindow Man, etc. Sphagnum workss constitutes of root, bunchs of subdivisions and little foliages, normally it incorporate both populating cells ( Chlorophyllose cells ) and dead cells ( Hyaline cells ) with capacity of keeping H2O in big sums. The peat moss peat moss can be easy distringwished from the other mosses by its alone subdivisions. Northern Hemisphere is the majority beginning for the peat mosses, different sort of species available over there and besides present in the South Hemisphere but found less species because of the handiness of more lake and humid weather conditions.

Lange and Jensen already reported a study about the spread of mosses in the Greenlands ( 1887 ) and the inside informations of sphagna was given by Jensen ( 1898, 1906, 1910 ) , and Harmsen and Seidenfaden ( 1932 ) , Harmsen ( 1933 ) and Hesselbo ( 1948 ) and the writers reports that the handiness of assorted species in different parts, it is chiefly reported from the Northern Hemisphere ( Bodil Lange ) . Sphagnum by and large finds on the wet countries chiefly where the H2O content is more. It is besides observed on the walls where wet or wetness is more and in the oozes and H2O pools. Particularly where the rainfall is more for illustration Scotland provides an ideal status for their growing. It is common in the northern Eurasia and northern North America due to more wet countries. In the Northern Hemisphere the peat moss population available extremely and are chiefly located I archipelago of Svalbard, Norway of Arctic at 81o N. In the Southern Hemisphere the population of peat moss found in New Zealand, Tasmania, Chile and Argentina.

Taxonomy of peat moss

Sphagnum genus is present in mosses runing from 100 to 300. Sphagnum has broad diverseness in the natural ecological system. There was figure of types of peat moss species are already listed, but it is still non complete. The genus Sphagnum classified depends on different classs, chiefly harmonizing to their morphological, chemical, size, colour and other characters.

Table 1: List of few peat moss species

Sl.no.

Division

List of species

1

Sphagnum affine

Sphagnum antarense

Sphagnum apiculatum

Sphagnum angustifolium

Sphagnum auriculatum

Sphagnum austinii

Sphagnum balticum

Sphagnum capillifolium

Sphagnum cristatum

Sphagnum compactum

Sphagnum cuspidatum

Sphagnum girgensohnii

Sphagnum humile

Sphagnum junghuhnianum

Sphagnum laricinum

Sphagnum magellanicum

Sphagnum majus

Sphagnum mendocinum

Sphagnum novo-caledoniae

Sphagnum novo-guineense

Sphagnum palustre

Sphagnum papillosum

Sphagnum perichaetiale

Sphagnum sedoides

These species have different type of characters. They normally have different forms, sizes of foliages, alone distribution of subdivisions, assorted types of macro characters and living nature.

Microbial activity of the genus Sphagnum

Sphagnum mosses are leftovers to decomposition ; it has compounds composed of curious chemical science. Sphagnum litter is composed of the specific secondary compounds such as polyphenols and uronic acid ; they are immune to the bugs and protect peat moss from decomposition. Because of its anti-microbial activity in antediluvian times it was used to continue the dead organic structures of the worlds, animate beings, etc.

Applications of the peat moss

Sphagnum has long term history more than 10,000 old ages of utilizations for worlds, animate beings and workss because of its natural home grounds. Due to its H2O keeping nature peat moss is used as dirt additive and besides sale as a dirt foil, it has possible to better productiveness in flaxen dirt and to better the agricultural productiveness ( Hong Li et al. , 2004 and Pulleman et al. , 2000 ) . Sphagnum is used as medicinally since 11th century because of its anti-microbial activity ( A.P. Podterob and E.V. Zubets ) . In Europe it has been used in the patchs for the lesion dressing and abscesses since. Because of the dry peat moss has absorptive nature and besides used as antiseptic, at the clip of first and 2nd universe war it was used for wound dressings and besides for bedding and babes diapers. The fume of the peat moss is used while fixing Scots whiskey and to give favour to it. Currently peat moss workss are used for the horticulture. In Roman times peat moss was used as a fuel, to cover the top of the houses. Due to its absorptive nature particularly it is used as oil absorbent to take oil spills. In United states its used as unwritten hygienic merchandise due to its absorbing and disinfectant nature for the tooth paste readyings as a bacterial vegetation in the unwritten pit of oral cavity ( A. P. Podterob and E. V. Zubets, 2002 ) . The species Sphagnum cristatum and Sphagnum subnitens are harvested in New Zealand are exported internationally for the usage of hanging basket line drives and for orchids as turning addendum. It is besides used as bedding stuff for the animate beings.

Polyphenols in peat moss

Polyphenols are the secondary compounds besides said to be defensive compounds ( H & A ; auml ; ttenschwiler and Vitousek, 2000 ) . Sphagnum is a rich beginning for phenolic compounds that act both as structurally and as inhibitors of microbic decomposition of the organic affair ( Verhoeven and Liefveld 1997 ; Freeman et al. 2001 ) . Lignin non present in Sphagnum ( Mauseth, 1998 ) , it contain other phenypropanoids as trans-sphagnum acid in majority ( Rasmussen et al. 1995 ) . Trans-sphagnum acid ( -p-hydroxyl-fl- [ carboxymethyl ] -cinnamic acid ) is derived from the cinnamic acid unique in peat mosses. Cinnamic acid is the cardinal compound for the synthesis of secondary polyphenols in peat moss it derived from the phenylalanine. The trans-sphagnum acid is synthesized by phenylpropanoid tract. The secondary metabolites produced in the phenylpropanoid tract may include flavonoids, hydroxycinnamic acid ester etc. The analysis of the bog H2O shows important sum of trans-sphagnum acid excreted from the peat moss, it is about 1µM ( Wilschke et al. , 1989 ) , so it may be necessary to analyse the sum of phenolic compounds persist in the cell wall and their elimination rate. In peat moss the incorporation of phenolic compounds mask the histochemical designation of cellulose and the in other side the Million ‘s reagent stains the cell wall in to ruddy colour. It proves that the peat moss species do non incorporate lignin as like higher workss.

Consequence of N on polyphenols

Nitrogen is the main food, of import for the workss and it controls the works growing. Both PAL1 and PAL2 show the response due to nitrogen and temperature. Surveies shows that depletion of the temperature and N degrees leads to double addition in PAL1 and PAL2 transcripts. It makes a anticipation that increasing degrees of N in the peat moss leads to diminishing the concentration of polyphenols. The peat moss bogs get their nutrition from the ambiance ; it creates an hypothesis that increasing degrees of the N in the ambiance may change the chemical science of the litter and C balance globally. Nitrogen is responsible for the amino acids and nucleotide formation ( Sittt and Krapp, 1999 ) . The changes in the C and nitrogen metamorphosis may alter the phenylpropanoid tract. In the add-on of N to the nitrogen-depleted workss shows change in the look of 100s of names ( Scheible et al. , 1997a, 2000 ; Scheible et al. , 2004 ; stitt, 1999 ; Wang et al. , 2000, 2003, 2004 ) . Polyphenols has strong repressive consequence against the bug, the increasing handiness of atmospheric N favours for microbic metamorphosis and speed up the decomposition of peat moss or litter. But, it leads to the emanation of higher sum of C dioxide ( CO2 ) and other C related merchandises later causes planetary heating.

Pathway of polyphenol synthesis in peat moss ( Phenylpropanoid pathway )

Phenolic compounds are alone in all peat mosses but the concentration of trans-sphagnum acid varies from the species ( Rudolph and Samland. , 1985 ) . Phenylpropanoids are organic compounds synthesized from cinnamic acid obtained from the phenylalanine in presence of the enzyme phenylalanine ammonia-lyase ( PAL ) . Phenypropanoid tract is alone in all vascular workss ( Lewis and Yamamoto, 1990 ) . Wide ranges of metabolites are produced in this tract and they are lending their consequence in works defence mechanism ( Nicholson and Hammerschmidt, 1992 ) and other environmental emphasiss. The compound include lignins, flavonoids, isoflavonoids and phytoalexins. 4CL ( 4-coumarate: CoA ligase ) is an ester derived from cinnamate ( Klaus Hahlbrock and Dierk Scheel. , 1989 ) . Cinnamic acid is the cardinal compound for the synthesis of secondary metabolites in all the workss. The polyphenols produced after a series of reactions as hydroxylation, methylation and desiccation and produces pCumaric acid, caffeic acid, ferulic and sinapic acid and simple coumarins. Phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase are the cardinal enzymes in the phenylpropanoid tract to bring forth secondary metabolites.

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Figure 4: The general tract of polyphenols in workss ( Phenylpropanoid pathyay )

Phenylalanine ammonium hydroxide lyase ( PAL )

It is normally present in all the green workss and it is the first enzyme involved in the phenylpropanoid tract. It ‘s an of import enzyme and most studied enzyme for the secondary metamorphosis in phenylpropanoid tract. It is the precursor for the major components, it converts phenylalanine in to trans-cinnamic acid in peat moss.

Cinnamic acid 4-hydroxylase

Cinnamate 4-hydroxylase belongs to cytochrome P450 monooxygenase, it invalves in the first measure of oxidization in the phenylpropanoid tract, it catalyzes the oxidation measure and plays cardinal function in the synthesis assortment of secondary metabolites. It is the 2nd enzyme involved in the phenylpropanoid tract. It converts trans-sphagnum acid in totrans-p-coumaric acid and besides controls the fluctuation of the C and protects the works from pathogens and UV radiation. C4H chiefly produces in response to visible radiation and works defence province like injuring and infections. The compounds formed due to C4H mechanism are indispensable for lignification and defence mechanism

Genes commanding the Phenylpropanoid tract in peat moss

Assorted cistrons commanding the enzymes involved in the phenypropanoid tract. It is possible to analyse the cistrons by sublimating the enzymes and cloning the several cistrons. These cistrons already isolated fom assorted works beings. The enzymes involved in the phenypropsnoid tract of peat moss are phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, trans-sphagnum acid synthase. Except the enzyme trans-sphagnum acid synthase the other enzymes are already studied in different workss for illustration Arabidopsis. PAL and C4H cistrons are of import and they code the cardinal enzymes phenyalanine ammonia lyase and cinnamic acid 4-hydroxylase. The FAH1 and F3H cistrons are responsible for the synthesis of flavonoids, they codes for the ferulate-5-hydroxylase and flavanone-3-hydroxylase. The PAL, C4H and flavonoid cistrons are produce in response to biotic or abiotic emphasiss, temperatures and external environmental factors. The cistrons PAL1 and PAL2 produce response to N and temperature, depletion of these environmental factors increases the PAL1 and PAL2 transcripts. To understand much about the function of PAL names in the ordinance of phenylpropanoid tract the PAL cistron from Gallic been ( Phaseolus vulgaris ) introduced into the baccy and studied the effects of the enzyme and its overexpression ( Elkind et al. , 1990 ) . It shows reduced endogenous PAL cistrons look in both RNA and enzymatic activity its due to feel suppression. The two cistrons C4H and ferulate-5-hydroxylase are hard to take due to the protein unstability. The cistron coding for the C4H was recognized ( Fahrendorf and Dixon, 1993 ; Mizutani et al. , 1993 ; Teutsch et al. , 1993 ) and purified the enzyme ( Gabriac et al. , 1991 ; Mizutani et al. , 1993 ) and the cistron encoding for ferulate-5-hydroxylase was cloned by labeling T-DNA ( Meyer et al. , 1996b ) .

The complementary DNA of the C4H cistron was isolated utilizing rearward transcriptase-PCR and RACE-PCR ( Rapid elaboration of the complementary DNA ends PCR ) from Korean native bramble ( Rubus coreanus Mique ) . In Arabidopsis complementary DNA isolated for a cytochrome P450, cinnamate 4-hydroxylase utilizing C4H complementary DNA of mung bean as a investigation. However the public presentation of C4H look with other names involved in phenylpropanoid tract is still unknown. The sequence analysis of 5 ‘ booster part of the C4H cistron ringer realizes that the look of PAL and 4CL cistrons is regulated due to the cis-elements involved in this procedure ( Longmann et al. , 1995 ) besides be present in the C4H booster.

Over look of PAL and C4H cistrons

The overexpression of PAL cistrons reported in different workss. The enzyme L-phenylalanine ammonia-lyae was overexpressed in the transgenic baccy contaiing PAL2 cistrons of been shows hushing of the endogenious PAL cistrons of baccy ( Paul A. Howles et al. , 1996 ) . In the trans genetic roots of C.blumei incorporating the Arabidopsis thaliana PAL1 cistron, and commanding under CaMV 35S booster shows the take downing the activity of phenylalanine ammonium hydroxide lyase and besides decreses the sum of phenoplasts, rosmarinic acid and chlorogenic acid. The F5H overexpression in Arabidopsis under control of cinnamic acerb 4-hydroxylase as apromoter shows important alterations in F5H look ( Knut Meyer et al. , 1997 ) . In Arabidopsis the C4H look was analyzed utilizing RNA smudge hybridisation, it shows modest addition in C4H look ( Dolly A. Bell-Lelong et al. , 1997 ) . The above groundss making an hypothesis that the complete look of PAL and C4H cistrons in peat moss may change the phenylpropanoid tract and possibly diminish the production of the phenolic compounds

Arabidopsis as a theoretical account species for peat moss

Arabidopsis is a theoretical account works for figure of researches, it is a little and flowering works and contains smallest cistron. Because of its broad scope of surveies it is known as theoretical account being for assorted types of cistron related surveies. To understand the assorted cistron related issues in peat moss, Arabidopsis is taken as a theoretical account, because it is the being of pick. The program Arabidopsis provides a planetary cistron look informations because it exposed to assorted interventions include light, temperature and salt or desiccation. The meta-analysis of the Arabidopsis provides possible offers to find cellular procedure and its associated cistrons and besides unknown cistrons. The phenylpropanoid tract in Arabidopsis contains three fluorescent sinapate esters ( sinapoylglucose, sinapoylmalate and sinapoylcholine ) . In arabidopsi the phenyalanine ammonia-lyase is coded by four cistrons. They are PAL1, PAL2, PAL3 and PAL4. The PAL1 and PAL2 contains about indistinguishable cryptography part, but in PAL3 and PAL4 it shows less similarity ( E Logemann et al. , ) . The quantitative existent clip PCR surveies shows the alterations in C $ H cistron forms ( Baek MH et al. , 2008 ) .

Brief experimental protocol

Sphagnum is an of import works in the natural ecosystem and it is necessary to understand the familial building and alterations to look into polyphenols in it. In order to understand about the familial control of the phenotype, it is necessary to foretell or hypothesis can be tested, so in the fallowing experiment propose to probe by the hypothesis ( based on the familial concept ) .

From the above inside informations PAL and C4H cistrons are of import in phenylpropanoid tract, these cistrons already cloned and sequenced from Arabidopsis thaliana. In the experiment we will utilize the coding sequence of these to insulate homologues from peat moss. We ab initio fallow the known Arabidopsis cistrons to seek for matching homologues sphagnum utilizing southern smudge. It will let us to gauge the figure of peat moss cistrons crossbreeding to the Arabidopsis cistron sequences, their transcript and approximative size of them. Then we will utilize the pervert primers constructed for Arabidopsis PAL and C4H cistrons and PCR to magnify the corresponding peat moss homologues, so which will cloned into the plasmid or phage vectors. These cloned cistrons will be sequenced, and used in northern hybridisations on peat moss to assay for the look profile of these cistrons. And at this phase we will hold isolated sphagnum cistrons involved in the synthesis of trans-sphagnum acid and find their look profile. In this experiment we will utilize a new cloning technique that is The TOPO® Cloning engineering

The TOPO® Cloning engineering

The enzyme DNA topoisomerase I is the cardinal cardinal enzyme in this engineering, it works as a limitation enzyme and as a ligase. The rule involved that the pentameric sequences 5′- ( C/T ) CCTT-3 ‘ is recognized particularly by Vaccinia virous topoisomerase I and organize the covalent bonds with phosphate group attached to the 3 ‘ thymidine and cleaves one Deoxyribonucleic acid strand leads to wind off the Deoxyribonucleic acid. To adhere the transportation activity of topoisomerase, TOPO® vectors are provided linearized with topoisomerase I covalently bound to each 3? phosphate. This enables the vectors to readily ligate DNA sequences with compatible terminals. The ligation is complete in merely 5 proceedingss at room temperature ( Suzanne M. Paquette et al. , May 2000 ) .

The cardinal aims of this experiment

To stabilise the concentration of the nursery gas in the ambiance by suppressing the release of locked C from the peat moss.

Bodil Lange. The genus peat moss in Greenland, June-1952, the Bryologist. Vol.55. No.2.

A.P. Podterob and E. V. Zubets. A History of Medicinal Use of Plants of Genus Sphagnum. Medical Plants, Pharmaceutical Chemistry Journal, Vol. 36, No. 4, 2002.

Suzanne M. Paquette, S & A ; oslash ; ren Bak, Ren & A ; eacute ; Feyereisen. Deoxyribonucleic acid and Cell Biology. May 2000, 19 ( 5 ) : 307-317.

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