Iyo pine nematode inzvimbo yakavharirwa inotama-endoparasite inozivikanwa nekukonzera kurasikirwa kwakanyanya kwehupfumi mumasango epine ecosystem. Chidzidzo chezvino chinoongorora chiitiko chenemacidal che halogenated indoles kurwisa pine nematodes uye maitiro avo ekuita. Iyo nemacidal zviitiko zve 5-iodoindole uye avermectin (yakanaka kutonga) kurwisa pine nematodes yaive yakafanana uye yakakwirira pane yakaderera yekuisa (10 μg / mL). 5-iodoindole yakaderedza fecundity, basa rekubereka, kufa kwe embryonic uye larval, uye locomotor maitiro. Molecular interactions of ligands ne invertebrate-specific glutamate-gated chloride channel receptors inotsigira pfungwa yokuti 5-iodoindole, kufanana neavermectin, inosunga zvakasimba kune receptor inoshanda nzvimbo. 5-Iodoindole yakakonzerawo zvakasiyana-siyana phenotypic deformations mune nematodes, kusanganisira abnormal organ kudonha/shrinkage uye kuwedzera vacuolization. Mhedzisiro iyi inoratidza kuti vacuoles vanogona kuita basa mukufa nematode methylation-mediated. Zvakakosha, 5-iodoindole yakanga isiri-chepfu kune ese marudzi ezvirimwa (kabichi neradish). Nekudaro, chidzidzo ichi chinoratidza kuti iodoindole application pasi pemamiriro ekunze inogona kudzora kukuvara kwepine wilt.
Pine wood nematode (Bursaphelenchus xylophilus) ndeye pine wood nematodes (PWN), migratory endoparasitic nematodes inozivikanwa nekukonzeresa zvakanyanya kukuvadza kwe ecological ku pine forest ecosystems1. Chirwere chepine wilt (PWD) chinokonzerwa nepine wood nematode chave kunetsa zvakanyanya mumakondinendi akati wandei, kusanganisira Asia neEurope, uye kuNorth America, nematode inoparadza pine marudzi akaunzwa1,2. Kuderera kwemuti wepaini idambudziko guru rehupfumi, uye tarisiro yekupararira kwayo pasi rose iri kunetsa3. Mhando dzepaini dzinotevera dzinonyanya kurwisa nematode: Pinus densiflora, Pinus sylvestris, Pinus thunbergii, Pinus koraiensis, Pinus thunbergii, Pinus thunbergii, uye Pinus radiata4. Pine nematode chirwere chakaipisisa chinogona kuuraya miti yemupaini mukati memavhiki kana mwedzi yehutachiona. Pamusoro pezvo, kubuda kwepine nematode kwakajairika munzvimbo dzakasiyana siyana, saka cheni dzehutachiona dzinoramba dziripo dzakagadzirwa1.
Bursaphelenchus xylophilus is a quarantine plant-parasitic nematode ye superfamily Aphelenchoidea uye clade 102.5. Iyo nematode inodya fungi uye inobereka muhuni yemiti yepine, ichikura kuva nhanho ina dzakasiyana dzemagonye: L1, L2, L3, L4 uye munhu mukuru1,6. Mumamiriro ezvinhu ekushomeka kwechikafu, pine nematode inopinda mune yakasarudzika dauer dauer, iyo parasitizes vector yayo - pine bark beetle (Monochamus alternatus) uye inotamisirwa kune ine hutano pine miti. Mune mauto ane hutano, nematodes inokurumidza kutama kuburikidza nemiti yemiti uye inodya parenchymatous masero, izvo zvinotungamirira kuhuwandu hwekunzwa kwe hypersensitivity, pine wilting uye kufa mukati megore mushure mekutapukirwa1,7,8.
Kudzora kwebiological pine nematodes kwave kunetsa, nematanho ekuvharirwa anotangira muzana ramakore rechi20. Nzira dzazvino dzekudzora pine nematodes dzinonyanya kusanganisira kurapwa kwemakemikari, kusanganisira kupfuta kwehuni uye kuisirwa nematicides muhunde dzemiti. Nematicides inonyanya kushandiswa ndeye avermectin uye avermectin benzoate, iyo ndeyemhuri yeavermectin. Makemikari anodhura aya anoshanda zvakanyanya kurwisa mhando zhinji dzenematode uye anoonekwa seakachengeteka kwezvakatipoteredza. Nekudaro, kudzokororwa kushandiswa kweaya nematicides kunotarisirwa kuumba kudzvanywa kwekusarudza izvo zvinozotungamira mukubuda kweanopokana nemipine nematodes, sezvakaratidzwa kune akati wandei tupukanana, seLeptinotarsa decemlineata, Plutella xylostella uye nematodes Trichostrongylus colubriformis uye Ostertagia vertagita 1 zvishoma nezvishoma yakagadzira circumcinins12. Naizvozvo, maitiro ekupikisa anofanirwa kugara achidzidzwa uye nematicides inoongororwa nguva nenguva kuti uwane imwe nzira, inodhura-inoshanda uye inochengetedza nharaunda matanho ekudzora PVD. Mumakumi emakore achangopfuura, nhamba yevanyori vakakurudzira kushandiswa kwemiti yemiti, mafuta anokosha uye mavhiriki semamatode control agents13,14,15,16.
Isu munguva pfupi yapfuura takaratidza basa remacidal re indole, intercellular uye interkingdom inoratidzira molecule, muCaenorhabditis elegans 17. Indole inopararira intracellular chiratidzo mu microbial ecology, inodzora mabasa akawanda anobata microbial physiology, spore formation, plasmid kugadzikana, kusagadzikana kwezvinodhaka, biofilm formation, uye virulence 18, 19. Basa re indole uye zvinobva kune mamwe pathogenic nematodes harina kudzidza. Muchidzidzo ichi, takaongorora chiitiko chenemacidal che34 indoles kurwisa pine nematodes uye tikajekesa magadzirirwo ekuita kwe5-iodoindole ine simba zvakanyanya tichishandisa microscopy, kutora-nguva-kupfura mafoto, uye ma molecular docking kuyedza, uye tikaongorora mhedzisiro yayo muchetura pazvirimwa uchishandisa ongororo yekumera kwembeu.
High concentrations (> 1.0 mM) yeindole yakambotaurwa kuti ine nematicidal effect panematodes17. Kutevera kurapwa kweB. xylophilus (matanho ehupenyu akavhenganiswa) ne indole kana 33 yakasiyana-siyana yeindole derivatives pa 1 mM, kufa kweB. xylophilus kwakayerwa nekuverenga nematode mhenyu uye yakafa mukutonga uye mapoka anorapwa. Indoles shanu dzakaratidza chiitiko chakakosha nematicidal; kupona kweboka risina kubatwa rinodzora raive 95 ± 7% mushure memaawa makumi maviri nemana. Pakati pe34 indoles yakaedzwa, 5-iodoindole uye 4-fluoroindole pa1 mM yakakonzera kufa kwe100%, asi 5,6-difluoroindigo, methylindole-7-carboxylate, uye 7-iodoindole yakakonzera inenge 50% kufa (Table 1).
Mhedzisiro ye5-iodoindole pakugadzira vacuole uye metabolism yepine huni nematode. (A) Mhedzisiro yeavermectin uye 5-iodoindole pavarume vakuru nematodes, (B) L1 danho nematode mazai uye (C) metabolism yeB. xylophilus, (i) vacuoles havana kuonekwa pa 0 h, kurapwa kwakaguma (ii) vacuoles, (iii) kuunganidza kwevacuoles yakawanda, vacuoles, yevacuoles, (iv) uye (vi) kuumbwa kwemahombe vacuoles. Miseve mitsvuku inoratidza kuzvimba kwevacuoles, miseve yebhuruu inoratidza kusanganiswa kwevacuoles uye miseve nhema inoratidza giant vacuoles. Scale bar = 50 μm.
Mukuwedzera, chidzidzo ichi chakatsanangurawo kutevedzana kwemethane-induced death mupine nematodes (Mufananidzo 4C). Methanogenic death is a non-apoptotic type of cell death inobatanidza nekuunganidzwa kweakakurumbira cytoplasmic vacuoles27. Izvo zvirema zve morphological zvinoonekwa mupine nematodes zvinoratidzika kunge zvine hukama nemaitiro ekufa kwemethane-induced death. Kuongororwa kweMicroscopic panguva dzakasiyana kwakaratidza kuti giant vacuoles akaumbwa mushure memaawa makumi maviri ekuratidzwa kune 5-iodoindole (0.1 mM). Microscopic vacuoles yakaonekwa mushure me8 h yekurapa, uye nhamba yavo yakawedzera mushure me12 h. Mazhinji mavacuoles makuru akaonekwa mushure me14 h. Mazhinji fused vacuoles ainyatsooneka mushure me12-16 h yekurapa, zvichiratidza kuti vacuole fusion ndiyo hwaro hwemethanogenic yekufa nzira. Mushure memaawa makumi maviri, mahombe mahombe akawanda akawanikwa muhonye yose. Aya maonero anomiririra mushumo wekutanga we metuosis muC. elegans.
Mune 5-iodoindole-yakabatwa nehonye, vacuole aggregation uye kuputika zvakaonekwawo (Fig. 5), sezvinoratidzwa nehonye inokotama uye vacuole kusunungurwa munzvimbo. Kuvhiringidzwa kweVacuole kwakaonekwawo mu membrane yezai, iyo inowanzochengetedzwa yakasimba neL2 panguva yekuvhara (Supplementary Fig. S2). Aya maonesheni anotsigira kubatanidzwa kwemvura kuunganidza uye kukundikana kwe osmoregulatory, pamwe nereversible cell injury (RCI), mukugadzirisa vacuole kugadzirwa uye suppuration (Fig. 5).
Tichifungidzira basa reiodine mune yakacherechedzwa kuumbwa kwevacuole, isu takaongorora nematicidal chiitiko che sodium iodide (NaI) uye potassium iodide (KI). Zvisinei, pazvikamu (0.1, 0.5 kana 1 mM), hazvina kukanganisa kana nematode kupona kana kuumbwa kwevacuole (Supplementary Fig. S5), kunyange zvazvo 1 mM KI yakanga ine zvishoma nematicidal effect. Kune rumwe rutivi, 7-iodoindole (1 kana 2 mM), kufanana ne5-iodoindole, yakakonzera vacuoles akawanda uye deformations yemaitiro (Supplementary Fig. S6). Iwo maviri iodoindoles airatidza akafanana phenotypic maitiro mupine nematodes, nepo NaI neKI vasina. Zvinofadza kuti indole haina kukonzera vacuole kugadzirwa muB. xylophilus pazviyero zvakaedzwa (data isina kuratidzwa). Nokudaro, zvigumisiro zvakasimbisa kuti indole-iodine complex ndiyo inokonzera vacuolization uye metabolism yeB xylophilus.
Pakati pemaindoles akaedzwa nematicidal basa, 5-iodoindole yaiva nepamusoro-soro slip index ye -5.89 kcal / mol, inoteverwa ne7-iodoindole (-4.48 kcal / mol), 4-fluoroindole (-4.33), uye indole (-4.03) (Mufananidzo 6). Iyo yakasimba musana wehydrogen bonding ye5-iodoindole kune leucine 218 inodzikamisa kusunga kwayo, nepo zvimwe zvese zvinobva muindole zvinosungirira ku serine 260 kuburikidza nedivi cheni hydrogen bond. Pakati peimwe iodoindoles inofananidzwa, 2-iodoindole ine kukosha kwekusungirirwa kwe -5.248 kcal / mol, iyo inokonzerwa nehrojeni yayo huru yekubatanidza ne leucine 218. Zvimwe zvinozivikanwa zvinosungirirwa zvinosanganisira 3-iodoindole (-4.3 kcal / mol), 4-iodoindole (-4.0) uye 6. kcal / mol) (Supplementary Figure S8). Mazhinji halogenated indoles uye indole pachayo, kunze kwe5-iodoindole uye 2-iodoindole, inoumba chisungo ne serine 260. Ichokwadi chokuti hydrogen bonding ne leucine 218 inoratidza kushanda kwe-receptor-ligand kusunga, sezvinoonekwa kune ivermectin (Supplementary Fig.5) Fig. 2-iodoindole, kufanana nevermectin, sunga zvakasimba kune nzvimbo inoshandiswa yeGluCL receptor kuburikidza neleucine 218 (Fig. 6 uye Supplementary Fig. S8). Isu tinokurudzira kuti kusungirirwa uku kunodiwa kuchengetedza yakavhurika pore chimiro cheGluCL yakaoma uye kuti nekusunga zvakasimba kune inoshanda saiti yeGluCL receptor, 5-iodoindole, 2-iodoindole, avermectin uye ivermectin saka chengetedza ion chiteshi chakavhurika uye kubvumira kunyura kwemvura.
Molecular docking ye indole uye halogenated indole kuGluCL. Kusunga maitiro e (A) indole, (B) 4-fluoroindole, (C) 7-iodoindole, uye (D) 5-iodoindole ligands kunzvimbo inoshanda yeGluCL. Iyo puroteni inomiririrwa neribhoni, uye musana wehydrogen zvisungo zvinoratidzwa seyero dotted mitsetse. (A′), (B′), (C′), uye (D′) anoratidza kudyidzana kwemarunda anofambirana neakatenderedza amino acid zvisaririra, uye eparutivi-cheni yehydrogen bond anoratidzwa nepingi ine miseve ine madoti.
Maedzo akaitwa kuti aongorore muchetura we5-iodoindole pakumera kwekabhichi nembeu dzeradish. 5-iodoindole (0.05 kana 0.1 mM) kana avermectin (10 μg/mL) yakanga ine zvishoma kana kuti isina mhedzisiro pakutanga kumera uye kubuda kwechirimwa (Mufananidzo 7). Pamusoro pezvo, hapana mutsauko wakakosha wakawanikwa pakati peyero yekumera yezvinodzora zvisina kurapwa uye mbeu inobatwa ne5-iodoindole kana avermectin. Mhedzisiro pakureba kwetaproot uye nhamba yemidzi yemashure yakaumbwa yakanga isingakoshi, kunyange zvazvo 1 mM (10 times its active concentration) ye5-iodoindole zvishoma yakanonotsa kukura kwe lateral midzi. Mhedzisiro iyi inoratidza kuti 5-iodoindole haina chepfu kukudyara maseru uye haikanganise maitiro ekukura kwechirimwa pane zvakadzidzwa.
Mhedzisiro ye5-iodoindole pakumera kwembeu. Kumera, kumera uye kudzika midzi kweB. oleracea neR. raphanistrum paMurashige neSkoog agar medium ine kana isina avermectin kana 5-iodoindole. Kumera kwakanyorwa mushure memazuva matatu ekuvharirwa pa22°C.
Chidzidzo ichi chinoshuma nyaya dzakawanda dzekuuraya nematode nema indoles. Zvakakosha, iyi ndiyo mushumo wekutanga weiodoindole inducing methylation (maitiro anokonzerwa nekuunganidza madiki mavacuoles anobatana zvishoma nezvishoma muhombe vacuoles, pakupedzisira zvichiita kuti membrane iparadze uye kufa) mumapine tsono, iodoindole ichiratidza zvakakosha nematicidal properties yakafanana neiyo yekutengesa nematicide avermectin.
Indoles yakambotaurwa kuti iite akawanda masaini mabasa muprokaryotes uye eukaryotes, kusanganisira biofilm inhibition / kuumbwa, kurarama kwebhakitiriya, uye pathogenicity19,32,33,34. Munguva pfupi yapfuura, zvinogona kurapa mhedzisiro yehalogenated indoles, indole alkaloids, uye semisynthetic indole zvinobva zvakwezva kutsvagurudza kufarira35,36,37. Semuenzaniso, halogenated indoles yakaratidzwa kuuraya inopfuurira Escherichia coli uye Staphylococcus aureus masero37. Pamusoro pezvo, zvine chekuita nesainzi kudzidza kushanda kwema halogenated indoles kurwisa dzimwe mhuka, genera, nehumambo, uye chidzidzo ichi inhanho yekuzadzisa chinangwa ichi.
Pano, tinokurudzira nzira ye 5-iodoindole-induced lethality muC. elegans inobva pa reversible cell injury (RCI) uye methylation (Mifananidzo 4C uye 5). Edematous shanduko dzakadai sekuzvimba uye vacuolar degeneration zviratidzo zveRCI uye methylation, inoratidzwa sehombe vacuoles mu cytoplasm48,49. RCI inokanganisa kugadzirwa kwesimba nekuderedza kugadzirwa kweATP, zvichikonzera kukundikana kwepombi yeATPase, kana kukanganisa maseru membranes uye kukonzera kukurumidza kupinda kweNa +, Ca2 +, uye mvura50,51,52. Intracytoplasmic vacuoles inomuka mumasero emhuka semugumisiro wekuunganidza kwemvura mu cytoplasm nekuda kwekuwedzera kweCa2 + uye mvura53. Zvinofadza kuti iyi nzira yekukuvara kwesero inogadziriswa kana kukanganisa kwekanguva uye masero anotanga kubudisa ATP kwenguva yakati, asi kana kukanganisa kunoramba kuripo kana kuwedzera, masero anofa.54 Zvatinoona zvinoratidza kuti nematodes inobatwa ne5-iodoindole haikwanisi kudzorera biosynthesis yakajairwa mushure mekusangana nemamiriro ekushungurudzika.
Iyo methylation phenotype inokonzerwa ne5-iodoindole muB. xylophilus inogona kunge yakakonzerwa nekuvapo kweiodine uye kupararira kwayo kwema molecular, sezvo 7-iodoindole yakanga ine zvishoma inhibitory effect paB. xylophilus kupfuura 5-iodoindole (Table 1 uye Supplementary Figure S6). Mhedzisiro iyi inoenderana nezvidzidzo zveMaltese et al. (2014), uyo akataura kuti kufambiswa kweiyo pyridyl nitrogen moiety muindole kubva kupara- kuenda kune meta-nzvimbo yakabvisa vacuolization, kukura inhibition, uye cytotoxicity mumasero eU251, zvichiratidza kuti kudyidzana kwemorekuru ine chaiyo inoshanda saiti muprotein yakakosha27,44,45. Kudyidzana pakati peindole kana halogenated indoles uye GluCL receptors yakaonekwa muchidzidzo ichi inotsigirawo pfungwa iyi, sezvo 5- uye 2-iodoindole yakawanikwa ichisunga kuGluCL receptors zvakanyanya kudarika mamwe ma indoles akaongororwa (Mufananidzo 6 uye Supplementary Figure S8). Iyo iodine panzvimbo yechipiri kana yechishanu yeindole yakawanikwa ichisunga kune leucine 218 yeGluCL receptor kuburikidza nemusana wehydrogen bonds, nepo mamwe halogenated indoles uye indole pachayo inoumba isina simba side-chain hydrogen bonds ne serine 260 (Mufananidzo 6). Saka isu tinofungidzira kuti nzvimbo yehalogen inobata basa rinokosha mukugadzirwa kwevacuolar degeneration, asi kusungirirwa kwakasimba kwe5-iodoindole kunochengetedza ion channel yakazaruka, nokudaro zvichibvumira kukurumidza kuyerera kwemvura uye kuputika kwevacuole. Nekudaro, iyo yakadzama nzira yekuita kwe5-iodoindole inoramba ichitemwa.
Pamberi pekushandisa kwe5-iodoindole, muchetura wayo pazvirimwa unofanirwa kuongororwa. Kuedza kwedu kumera kwembeu kwakaratidza kuti 5-iodoindole yakanga isina mhedzisiro yakaipa pakumera kwembeu kana nzira dzekusimudzira dzakazotevera pane zvakadzidzwa (Mufananidzo 7). Nokudaro, chidzidzo ichi chinopa hwaro hwekushandiswa kwe5-iodoindole munharaunda yezvakatipoteredza kudzora kukuvadza kwepine nematodes kumiti yemapine.
Mishumo yapfuura yakaratidza kuti indole-based therapy inomiririra nzira inogona kugadzirisa dambudziko rekuramba kwemishonga inorwisa mabhakitiriya uye kufambira mberi kwegomarara55. Mukuwedzera, indoles ine antibacterial, anticancer, antioxidant, anti-inflammatory, antidiabetic, antiviral, antiproliferative uye antituberculosis mabasa uye inogona kushanda sechinhu chinovimbisa chekugadzirwa kwezvinodhaka56,57. Ichi chidzidzo chinoratidza kwekutanga kushandiswa kunogona kushandiswa kweiodine se antiparasitic uye anthelmintic agent.
Avermectin yakawanikwa makumi matatu emakore apfuura uye yakahwina Mubairo weNobel muna 2015, uye kushandiswa kwayo seanthelmintic kuchiri kuenderera mberi. Zvisinei, nekuda kwekukurumidza kukura kwekupikisa avermectins mu nematodes uye zvipembenene zvipembenene, imwe nzira, yakaderera, uye yakagadzikana yezvakatipoteredza inodiwa kudzora hutachiona hwePWN mumiti yemapine. Ichi chidzidzo chinoshumawo magadzirirwo ayo 5-iodoindole inouraya pine nematodes uye kuti 5-iodoindole ine yakaderera muchetura kumaseru ekudyara, iyo inovhura tarisiro yakanaka yekushandiswa kwayo kwekutengeserana mune ramangwana.
Zvese zviedzo zvakatenderwa neEthics Committee yeYeungnam University, Gyeongsan, Korea, uye nzira dzakaitwa zvichiteerana negwara reEthics Committee yeYeungnam University.
Egg incubation experiments yakaitwa pachishandiswa nzira dzakatarwa43. Kuongorora mazinga ehatching (HR), 1-day-old nematodes yakakura (inenge 100 vakadzi uye 100 varume) vakaendeswa kuPetri ndiro dzine fungus uye dzakabvumirwa kukura kwe24 h. Mazai akabva aparadzaniswa uye akabatwa ne5-iodoindole (0.05 mM uye 0.1 mM) kana avermectin (10 μg/ml) sekumiswa mumvura isina tsvina yakasvibiswa. Izvi zvakamiswa (500 μl; mazai angangoita 100) zvakaendeswa kumatsime e24-tsime tishu tsika ndiro uye incubated pa22 °C. Nhamba dzeL2 dzakaitwa mushure memaawa makumi maviri nemana ekuputira asi dzakanzi dzakafa kana masero asina kufamba kana akakurudzirwa newaya yakanaka yeplatinum. Kuedza uku kwakaitwa mumatanho maviri, imwe neimwe iine kudzokorora katanhatu. Iyo data kubva kune zvese zviedzo zvakabatanidzwa uye zvakaratidzwa. Iperesenti yeHR inoverengerwa sezvinotevera:
Kufa kweLarval kwakaongororwa pachishandiswa nzira dzakambogadzirwa. Mazai eNematode akaunganidzwa uye maembryos akawiriraniswa nekuchochonywa mumvura isina tsvina yakanyungudutswa kuti ibudise L2 nhanho. Synchronized larvae (anenge 500 nematodes) akabatwa ne5-iodoindole (0.05 mM uye 0.1 mM) kana avermectin (10 μg/ml) uye akarerwa paB. cinerea Petri mahwendefa. Mushure memaawa makumi mana nemasere ekukuchidzira pa22 ° C, nematode dzakaunganidzwa mumvura isina kucheneswa uye yakaongororwa kuvepo kweL2, L3, uye L4 nhanho. Kuvepo kweL3 neL4 nhanho kwakaratidza shanduko yelarval, nepo kuvapo kweiyo L2 nhanho kwairatidza kusashandurwa. Mifananidzo yakatorwa pachishandiswa iRiS™ Digital Cell Imaging System. Kuedza uku kwakaitwa mumatanho maviri, imwe neimwe iine kudzokorora katanhatu. Iyo data kubva kune zvese zviedzo zvakabatanidzwa uye zvakaratidzwa.
Kuchetura kwe5-iodoindole neavermectin kumbeu kwakaongororwa pachishandiswa bvunzo dzekumera paMurashige neSkoog agar plates.62 B. oleracea neR. raphanistrum mhodzi dzakatanga kunyikwa mumvura isina tsvina yakasvibiswa kwezuva rimwe, yakagezwa ne 1 ml 100% sterilized % 5 ml ethanol (100% sterilized % 5% yekutengesa, 3% yesodium ethanol). hypochlorite) kwemaminetsi gumi nemashanu, uye yakashambidzwa kashanu ne 1 ml mvura isina utachiona. Mbeu dzakaomeswa dzakazotsikirirwa pamapuleti eagar ekumera ane 0.86 g/l (0.2X) Murashige neSkoog medium uye 0.7% bacteriological agar ine kana isina 5-iodoindole kana avermectin. Mahwendefa acho akazoiswa pa22 ° C, uye mifananidzo yakatorwa mushure memazuva matatu ekuiswa. Kuedza uku kwakaitwa mumatanho maviri, chimwe nechimwe chaive nekudzokorora katanhatu.
Nguva yekutumira: Feb-26-2025