Me yasa Babban Darasin Injiniya na Protein Ya Aiwatar da Ingantaccen Peptide
Filin peptide ya mamaye levers na gina jiki injiniya - cyclization, N-methylation, PEGylation, stapling - ba tare da ko da yaushe sha ta horo. Injiniyan sunadaran sun sami tsauri game da ciniki saboda ya zama dole: Fc-fusion ko ingantaccen haɓakar haɓaka yana da tsada don yin, jinkirin gwadawa, kuma sakamakon asibiti idan ya gaza. Wannan matsa lamba ya haifar da saitin halaye waɗanda ke canzawa kai tsaye zuwa aikin peptide, kuma su ne halaye iri ɗaya waɗanda ke raba haɓaka peptide daga jerin dabarun sinadarai.
Halaye hudu sun fi muhimmanci.
Na farko, Injiniyoyi sunadaran suna adana kayan aikin da injiniyoyin da ke kewaye da shi. Na biyu, suna aiwatar da ƙira mara kyau - zaɓin maye gurbin musamman don cire lamunin ɓarna da tarawa, ba kawai don ƙara alaƙa ba. Na uku, suna kula da masu haɗin gwiwa, masu sarari, da juzu'i na lissafi a matsayin abubuwa masu aiki. Na hudu, suna buƙatar shaida ta orthogonal cewa kwayoyin shine abin da suke tsammani shine. Kowane ɗabi'a ya zama darasi ɗaya a ƙasa.
Darasi 1: Kiyaye Pharmacophore Kafin Ka Daidaita Komai
Peptide Synthesis Mafi yawan gazawar da ake samu a inganta peptide shine maye gurbin "tsayawa" wanda ke share lamba mai mahimmanci a hankali.. Injiniyan FGF yana nuna yadda kunkuntar ta ke: Canjin S137P yana haɓaka kwanciyar hankali a cikin FGF2 ta hanyar daidaita heparin-daure β-hairpin ta hanyar tattarawar CH-π tsakanin P137 da zoben indole W123, yayin da ragowar da ke samar da haɗin mai karɓa dole ne su kasance ba a taɓa su ba (Binciken Tsarin Tsarin Halitta da Biochemical cikin Stable FGF2 Variants). Canjin daidaitawa yana aiki saboda yana ƙarfafa ɓarna, ba don yana sake rubuta saman dauri ba.
Hankali guda yana sarrafa aikin peptide. Kafin yin wani canji, ayyana ƙaramin motsin aiki - yawanci saura huɗu ko fiye - kuma ku bi da shi azaman kafaffen. bambance-bambancen FGF1 masu ƙarfi suna kwatanta horo a sikelin: ƙira irin su Q40P/S47I/H93G/S99A/K118E sun haɓaka kwanciyar hankali sosai yayin da suke riƙe aikin mitogenic da na rayuwa., saboda an zaɓi sauye-sauye masu daidaitawa don tasirin su akan tattarawa da juriya na proteolytic maimakon kusancin su zuwa wurin ɗaure. (Injiniya FGF1 Bambance-Bambance Tsakanin Ma'aurata Daga Ayyuka).
Ƙungiyar aiki mai amfani ita ce dangin analog. Gabatar da sauyi guda ɗaya wanda ke haifar da hasashe a lokaci guda - musanya guda ɗaya, takura guda daya, hula guda ɗaya - yana kiyaye kowane tasiri mai yiwuwa, kuma saitin analogs masu alaƙa da raba iyaye sun fi ba da labari fiye da tsarin ƙirar da ba su da alaƙa. Wannan ƙa'idar kuma ita ce ke jagorantar waɗanda 'yan takara suka cancanci haɗawa kwata-kwata: fifita waɗanne analogs sun cancanci haɗawa wani bangare ne na ingantawa kamar sinadarai da kanta.
Sa hannu na gazawar biyu ya cancanci a duba. Zumunci yana raguwa yayin da kowane ma'aunin kwanciyar hankali ya inganta, wanda yawanci yana nufin maye gurbin ya taɓa ragowar lamba, ya canza matsayin dauri, ko kuma takura peptide cikin lissafi mai karɓa ba zai iya shiga ba. Na biyu ya fi dabara: gubar ya yi kama da tsabta ta nazari amma yana rasa ƙarfi a cikin aikin tantancewar, saboda canjin ya adana jerin abubuwan yayin da yake canza yanayin yanayin halitta.
Tabbatarwa yana bi kai tsaye. Haɗa kowane karatun kwanciyar hankali tare da ma'aunin ɗauri kai tsaye - resonance na plasmon ko isothermal titration calorimetry - da karatun wayar salula mai aiki.. Mafi girman zafin jiki na narkewa ba shaida ce mafi kyawun ƙwayar cuta ba.
Darasi 2: Injiniya don Tafarkin Karɓar Mahimmanci, Ba "Kwarzo ba"
"Kwanciyar hankali" ba dukiya ɗaya ba ce. Aikin FGF2 ya rabu da rarrabuwa daga bayyanar zafi: maye gurbin D28E ya rage raguwa yayin shiri, matsala daban-daban daga daidaitawar β-hairpin da S137P ke magana. kwanciyar hankali Peptide yana buƙatar horo iri ɗaya na bincike, saboda rashin zaman lafiya, rashin kwanciyar hankali ta jiki, da kuma cirewar enzymatic kowanne yana buƙatar gyara daban.
Rage ragowar Asn da Gln a cikin abubuwan da aka fallasa, yankuna masu sassaucin ra'ayi suna samar da nau'ikan caji da bambance-bambancen tsari, kuma yana canza hydrophobicity, caji, da taro lokaci guda - wanda ke nufin yana iya zama marar ganuwa zuwa kashi mai tsafta amma a bayyane ga duban taro. (Dokokin Gudanarwa don Nazarin Magungunan Peptides). Tari yana bin wata hanya ta daban, gudana ta hanyar hydrophobic runs da β-sheet propensity maimakon ta kowane saura mai amsawa guda ɗaya. (Abubuwan Da Suka Shafi Kwanciyar Jiki na Peptide Therapeutics). Tsabtace furotin shine matsala ta uku kuma, kuma yana mayar da martani ga ilmin sunadarai daban-daban.
Sakamakon aikace-aikacen shi ne cewa levers na gama gari ba sa canzawa. Kowanne yana siyan kadara guda ɗaya kuma ya yi mata caji a wani wuri.
| Lever zane | Abin da yake saya | Abin da ya saba tsada |
|---|---|---|
| Yin hawan keke / stapling | Kwanciyar hankali, proteolytic juriya | Zai iya kulle juzu'i marasa bioactive; yana ƙara hadaddun roba |
| D-amino acid, abubuwan da ba na canonical ba | Rage sanin protease | Canje-canjen lambobin sadarwa; mafi wuya hali |
| N-methylation | Rage haɗin haɗin hydrogen, inganta permeability | Zai iya tarwatsa mahimmin hulɗar dauri; ƙarin kira mai buƙata |
| Tashar tasha | Exopeptidase juriya | Ƙananan kariya daga tsagewar ciki |
| PEGylation ko lipidation | Tsawon rabin rayuwa, mafi girma daukan hotuna | Asarar ayyuka daga kariya mai ƙarfi; kara iri-iri |
| Ƙara hydrophobic Peptides na roba saura | Zubar da ciki | Tari, gazawar narkewa, assay artifacts |
Ingantacciyar ingantawa shine ma'aunin ƙarfi, zaɓe, proteolytic kwanciyar hankali, narkewa, permeability, da haɓakawa, ana bin sawu tare maimakon kadara ɗaya a lokaci ɗaya (Daga Jagora zuwa Kasuwa: Hanyoyi na Chemical don Sauya Peptides zuwa Magunguna). Inda makasudin shine permeability na musamman, Makasudin shine taga sinadari na sinadarai maimakon matsakaicin lipophilicity - kewayon kusan cLogP 2 ku 5 tare da polarity na kashin baya da aka binne - saboda turawa lipophilicity mafi girma yana ba da damar cinikin riba don haɓaka haɗarin tarawa. (Peptides azaman Tsarin Tsarin Kwayoyin Halitta).
Me gazawa yayi kama. Nasarar dukiya guda ɗaya wanda ke lalata bayanin martaba: analog mai keken keke tare da kyakkyawan juriya na protease wanda baya ɗaure, ko analog mai lipidated tare da tsawon rabin rayuwa kuma babu ƙarfi. Dabarun haifar da tsauri suna ɗaukar takamaiman sigar wannan haɗarin, kuma ƙirar peptide stapled da cyclized yana biya ne kawai lokacin da aka tabbatar da ƙuntatawa a kan ƙayyadaddun kwayoyin halitta maimakon a ɗauka don adana shi.
Yadda ake tabbatar da shi. Gudanar da karatun tilasta-lalata a ƙarƙashin zafi, pH, oxidation, da damuwa mai haske, sannan tabbatar da hanyar da ke nuni da kwanciyar hankali wanda a zahiri ke warware abubuwan lalata da suka haifar. Har ila yau, haɗe-haɗen guduro ya cancanci a duba shi da wuri: jerin abubuwan da za a iya ganowa a sikelin ganowa na iya haifar da ƙarancin ɗanyen inganci da tsarkakewa mai ƙarfi da zarar sun tashi sama a sikelin..
Darasi 3: Bi da Linkers, Tags, da Geometry na Haɗawa azaman Aiki
Tsawon rabin rayuwa shine inda injiniyoyin peptide sukan gaji matsalar injiniyan furotin ba tare da saninsa ba. A cikin haɗin ginin, sanyawa na fusion da yanayin jerin mahaɗin suna da mahimmanci don kiyaye ayyukan peptide - ilmin sunadarai da ke aiki a cikin wani nau'i na lissafi na iya hana aiki a wani. (Dabarun Injiniyan Protein don Dorewar Ayyukan GLP-1).
Bambance-bambancen FGF suna yin ma'ana guda daga kishiyar shugabanci. FGF2-STAB mutum ne mai maki tara FGF2 mutant tare da narkar da yanayin zafi har zuwa 19 °C da a kasa dogara ga heparin don siginar ERK/MAP fiye da furotin na daji (Binciken Tsarin Tsarin FGF2-STAB). Hankalin ba shine mutant ya kawar da hulɗar haɗin gwiwar sa ba. Shi ne cewa ƙira ta sake daidaita dogara ga cofactor maimakon soke shi, kuma an sake auna yanayin siginar maimakon a ɗauka.
Peptide conjugation ya cancanci magani iri ɗaya. Wurin da aka makala, tsawon linker, kuma masana kimiyyar sararin samaniya suna tantance ko babban mai ɗaukar kaya yana kare fuskar dauri. Haɗin kai guda biyu tare da kaya iri ɗaya da masu haɗin kai iri ɗaya na iya bambanta sosai a cikin aiki kawai daga inda abin da aka makala ya sauka..
Me gazawa yayi kama. A conjugate tare da kyakkyawan bayyanar da rashin ƙarfi, ko fusion wanda mai ɗaukar hoto ya toshe epitope mai ɗaure a kaikaice. Rashin gazawa sau da yawa ba a iya gani a cikin tsabta ko duban taro, saboda kwayar halitta ita ce ainihin abin da aka ƙera - kawai ba ya aiwatar da manufa ɗaya.
Yadda ake tabbatar da shi. Tabbatar da wurin haɗin gwiwa da stoichiometry na nazari, sannan auna dauri da aiki don conjugate kanta maimakon cirewa daga peptide mara nauyi. Inda makasudin shine rabin rayuwa maimakon takamaiman aikin lissafi, PEGylation da tsawon rabin rayuwa dabaru suna bin ka'ida ɗaya: da tsawo sunadarai da abin da aka makala batu ne zane masu canji, ba marufi ba.
Darasi 4: Yi Tsarin Samfurin Peptide na Zane, Ba Ceto Late ba
Tsarin tsari shine inda cinikin da kuka jinkirta dawowa zai dawo. A peptide tare da mai kyau iko da kwanciyar hankali profile iya har yanzu kasa idan ta solubility, halin tarawa, ko oxidation hankali kawai ya zama dacewa a taro da gabatar da shirin a zahiri yana buƙata.
Wannan tambaya ce-matakin ƙira, ba matakin ci gaba ba. Kaddarorin da ke ƙayyade halayen ƙira - cajin net, hydrophobic rarraba, kasancewar ragowar abubuwan da ke da iskar oxygen, Adadin masu ba da gudummawar haɗin gwiwar hydrogen da aka fallasa - duk an saita su ta hanyar da kuka zaɓa. Yanke shawara tsakanin analogs guda biyu in ba haka ba yana da sauƙin sauƙi yayin da ƙirƙira ƙarfi yana ɗaya daga cikin ma'auni daga farkon..
Hukunce-hukuncen tsari da kansu suna iya yiwuwa. Haɗin kai ta hanyar haɗin gwiwar hydrophobic da electrostatic yana amsawa ga zaɓin surfactant da haɓaka pH. Ƙananan solubility yana amsawa ga daidaitawar pH, hadin kai, ko abubuwan haɓakawa. Rashin kwanciyar hankali a kusa da pH mai tsaka-tsaki wanda ke haifar da deamidation da isomerization yana amsawa ga sarrafa pH, da kuma ajiyar lyophilized a ƙasa da pH 6 a cikin m jihar iyaka deamidation. Ana sarrafa Oxidation ta hanyar sarrafa sararin samaniya, inert-gas handling, amber ko duhu ajiya, da nisantar buɗaɗɗen vial mai maimaitawa.
Inda har yanzu hanyar gudanarwa a bude take, yana siffanta dukkan burin ingantawa - GI kwanciyar hankali, dabarun gyara sinadarai, rashin tsarki, kuma buƙatun bioanalytical suna canzawa tare maimakon masu zaman kansu. Wannan haɗin gwiwar ya cancanci yin taswira da wuri, saboda yadda hanyar gudanarwa ke sake fasalin abubuwan ci gaban peptide shine ainihin nau'in ƙuntatawa wanda ke ƙayyade wane analog a cikin saiti shine ainihin jagorar.
Me gazawa yayi kama. Shirin yana zaɓar jagora akan ƙarfi da kwanciyar hankali, sa'an nan ya gano a lokacin da aka ƙirƙira cewa kwayoyin sun taru sama da taro mai amfani, ko kuma ba za a iya sarrafa bayanan iskar oxygen ɗin sa a cikin akwati da shirin ke buƙata ba.
Yadda ake tabbatar da shi. Solubility na allo da tarawa da wuri - kafin cikakken ingantawar gubar - a cikin pH da yanayi masu ban sha'awa waɗanda ke cikin iyaka.. Allon ƙira na sama ne azaman tacewa akan saitin analog, maimakon kasa a matsayin ceto da aka yi wa duk dan takarar da ya tsira da karfinsa.
Darasi 5: Daidaita Tabbatar da Nazari zuwa Haɗarin ƙira
Tabbacin nazari ba akwati ne na saki ba. Hanya ce ta hanyar da za ku gano ko kwayar halittar da kuka tsara ita ce kwayar halittar da kuke da ita - kuma yawancin ku injiniyoyin jeri., nasaba, ko yanayin bayan fassara, yawan wannan tambayar yana buƙatar amsoshi masu zaman kansu. Yin la'akari da tabbatarwa na nazari azaman fitarwar ƙira maimakon dubawa ta ƙarshe shine abin da ke kiyaye analog mai alƙawarin ci gaba akan fakitin bayanan da ba a karanta ba..
Hanyoyin orthogonal suna yin nasara saboda suna amsa tambayoyi daban-daban na gaske. Kashi mai tsafta yana tambaya ko samfurin yana nuna nau'in chromatographic guda ɗaya. Ba ya tambayar menene taro, wane nau'in girman nau'in da ke wanzu a cikin bayani, ko kuma tsarin ya tsira.
| Hanyar nazari | Tambaya ta amsa | Matsalolin da yake ganowa |
|---|---|---|
| Farashin RP-HPLC | Shin wannan nau'in chromatographic guda ɗaya ne? | Masu lalata, truncations, oxidation da deamidation kololuwa, zarewar tsari |
| LC-MS / LC-MS/MS | Shin taro da jeri ne abin da aka tsara? | bambance-bambancen sharewa ko tsawo, aikin gyara-site, proteolysis |
| SEC-MALS | Wane nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i)) a cikin bayani? | Tari, oligomers, gutsuttsura - tare da cikakkar taro maimakon fa'idar lokacin riƙewa |
| CD | Shin tsarin na biyu yana nan? | Rashin hasara, β-shafin riba, bayyanawa hade da tarawa |
| NMR | An kiyaye yanayin sinadarai na gida? | Amintaccen tsari, isomers, da dabara gyare-gyare da cewa matsawa taro kadan |
| Farashin SPR / ITC | Suna daure kinetics da thermodynamics cikakke? | Asarar dangantakar da babu tsafta da ke iya gani |
Wannan kuma shine inda tsammanin tsari ke haɗuwa tare da kyakkyawan aiki. EMA ta Jagora akan Haɓaka da Haɓaka Peptides na roba yana ba da shawarar yin amfani da aƙalla hanyoyi biyu na orthogonal don gano peptide a ƙayyadaddun bayanai da saki, tare da zaɓin haɗin da ake buƙata don tabbatar da jerin ba tare da wata shakka ba. Ina Q2(R2) yana fuskantar fata iri ɗaya daga ɓangaren tabbatarwa: Ana iya nuna ƙayyadaddun ƙayyadaddun ƙayyadaddun bayanai ta hanyar kwatanta sakamako da na daƙiƙa, hanya mai kyau mai kyau bisa ga wata ka'idar auna ta daban.
Doka mai amfani ita ce ƙaddamar da kunshin tabbatarwa zuwa haɗarin ƙira. Sauyawa guda ɗaya mai ra'ayin mazan jiya a cikin siffa mai kyau yana buƙatar ƙasa; gyare-gyaren wurare da yawa, a hawan keke, ko conjugation yana buƙatar ƙarin. Inda tarawa ya dace, chromatography mai girma-keɓanta tare da watsawar haske mai yawa resolves high-molecular-weight species that a purity value alone cannot distinguish from a resolved impurity.
A matsayin misali na aiki na abin da wannan yayi kama a aikace, fakitin tabbatarwa na orthogonal na nau'in MOL Canje-canje yana samarwa kowace kuri'a - juzu'i na HPLC don tsabta, Mass spectrometry don ainihi, da ƙarin hanyoyin kamar SEC-MALS ko NMR inda haɗarin ƙira ya ba su garanti, goyan bayan da ƙayyadaddun chromatograms na musamman da yawa fiye da ƙididdiga masu yawa - shine nau'in shaida da ke ba da damar shirin ya danganta bambance-bambancen tsari ga ainihin dalili.. Wannan ya fi mahimmanci inda shirin zai buƙaci a ƙarshe don kare daidaiton tsari-zuwa-tsari: gani peptide microheterogeneity fiye da kashi ɗaya mai tsabta nawa adadin kashi-yanki zai iya kuma ba zai iya gaya muku ba.
Me gazawa yayi kama. Siffa mai tsabta da ke kama da karɓuwa yayin da ainihin nau'in da ke cikin bayani shine jimillar, bambance-bambancen deamidation, ko isomer na sitiriyo. Batch yana sakewa kuma kimantawar ba ta cika aiki ba.
Yadda ake tabbatar da shi. Gina fakitin tabbatarwa daga lissafin haɗarin ƙira: ainihi da jeri ta taro da tandem mass spectrometry, tsafta da lalata ta hanyar ingantacciyar kwanciyar hankali mai nuna hanyar chromatographic, tarawa ta hanyar tushen girman girma, tsarin ta CD ko NMR lokacin da daidaituwar al'amura, da aiki ta hanyar ɗaure ko tantance tushen tantanin halitta.
Jerin Ingantaccen Aiki
Darussan da ke sama suna rugujewa zuwa madauki mai maimaitawa maimakon tsarin layi.
- Ƙayyade bayanin martabar manufa. Saita makasudin kadarorin - ƙarfi, proteolytic kwanciyar hankali, narkewa, permeability, tsawon lokacin fallasa - da kuma auna su don nuni da hanyar da aka nufa.
- Gyara pharmacophore. Gano mafi ƙarancin motsin motsi kuma kula da shi azaman takura, ba mai canzawa ba.
- Yi ƙananan, m canje-canje. Sauya ɗaya, mahaɗin ɗaya, takura daya, ko shafin da aka makala daya akan kowane analog, don haka tasirin yana iya ganowa.
- Triage da wuri kuma a cikin layi daya. Auna aiki, kwanciyar hankali, narkewa, da tarawa ba da daɗewa ba bayan zagaye na farko na kira maimakon bayan cikakken ingantawa. Haɓaka gazawar da wuri ya fi arha fiye da gano shi a lokacin tsarawa.
- Yi bincike kafin a gyara. Rarrabe lalacewar sinadarai daga rashin kwanciyar hankali daga sharewa, kuma zaɓi lever wanda ke magance babbar hanyar.
- Tabbatar da kai tsaye. Daidaita fakitin tabbatarwa zuwa haɗarin ƙira kuma tabbatar da asalin kwayar halitta, tsarki, girman jihar, da tsari kafin ciyar da shi. Samuwar Peptide
- Rufe madauki. Ciyar da sakamakon da aka auna baya zuwa zagaye na gaba na ƙirar analog maimakon fara sabon saitin hasashe. Haɓaka jeri yana da maimaitawa ta yanayi; kowane zagaye ya kamata ya ƙunsar sararin ƙirar maimakon sake kunna shi.
FAQ
Me yasa canji mai daidaitawa zai iya rage ayyukan peptide? Domin kwanciyar hankali da aiki galibi yanki ɗaya ne ke ɗaukarsa. Sauya S137P a cikin FGF2 yana haɓaka kwanciyar hankali ta hanyar ƙarfafa β-hairpin ta hanyar tattarawar gida., amma sauye-sauyen da suka faɗo kan saman da ke fuskantar mai karɓa suna canza lissafin dauri. Doka mafi aminci ita ce a daidaita ɓangarorin kuma a bar pharmacophore ba a taɓa shi ba, sa'an nan kuma sake auna aiki a ƙarƙashin damuwa maimakon a zafin jiki.
Shin kashi ɗaya cikin tsarki ya isa don tabbatar da peptide daidai ne? A'a. Siffar tsaftar chromatographic guda ɗaya ta amsa ko samfurin ya zama nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'ንንን nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'''in nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau`ikan nau`ikan nau`ikan nau`ikan nau`ikan»"_"_"_"_"_"_"_*_. Ba ya tabbatar da taro, jerin, Jihar tari, ko tsarin. EMA tana ba da shawarar aƙalla hanyoyi guda biyu don ganowa a lokacin saki, kuma hadarin ƙira ya kamata ya ƙayyade ko ana buƙatar tushen girma ko hanyoyin tsari.
Yaushe yakamata aikin tsarawa ya fara dangane da inganta jagoranci? Tun da farko fiye da yawancin shirye-shiryen sun ɗauka. Solubility, halin tarawa, oxidation hankali, kuma tasirin pH duk an saita su ta jeri da zaɓin gyare-gyare, don haka su ne sigogin ƙira kamar yadda suke da sigogi na ci gaba. Nuna su tare da na'urar analog ta farko yana hana zaɓin gubar da ba za a iya ƙirƙira ta a ma'auni mai amfani ba.
Matakai na gaba
Idan kana riƙe da saitin analog wanda yayi kama da ƙarfi akan dukiya ɗaya kuma mai rauni a ko'ina, tattaunawa mai amfani shine fasaha: wanda hanyar lalacewa ta mamaye, abin da kunshin tabbatarwa ya kamata ya ƙunshi, kuma ko saitin tantancewa na yanzu zai iya bambanta ingantaccen haɓakawa daga kayan aikin aunawa. Magana da hakan ta sabawa takamaiman jerin ku ita ce hanya mafi sauri zuwa jagora mai karewa.
