I-Propionic acid (PPA), i-arhente yokubola kunye ne-common diet additive, ibonakalisiwe ukuba ibangela uphuhliso olungaqhelekanga lwe-neurodevelopment kwiimpuku ezihamba nokungasebenzi kakuhle kwesisu, okunokubangelwa yi-gut dysbiosis. Kuye kwacetyiswa ukuba kubekho unxibelelwano phakathi kokuvezwa kwe-PPA yokutya kunye ne-gut microbiota dysbiosis, kodwa ayikaphandwa ngokuthe ngqo. Apha, siphande utshintsho oluhambelana ne-PPA kwi-gut microbiota composition olunokukhokelela kwi-dysbiosis. Ii-gut microbiomes zeempuku ezitya ukutya okunganyangwanga (n=9) kunye nokutya okutyebiswe yi-PPA (n=13) zalandelelaniswa kusetyenziswa i-long-range metagenomic sequencing ukuvavanya umahluko kwi-microbial composition kunye neendlela ze-bacterial metabolic. I-Dietary PPA yayinxulunyaniswa nokwanda kobuninzi be-taxa ebalulekileyo, kuquka ii-Bacteroides ezininzi, i-Prevotella, kunye ne-Ruminococcus species, amalungu azo ebekhe abandakanyeka kwimveliso ye-PPA. Ii-microbiomes zeempuku ezivezwe yi-PPA nazo zazineendlela ezininzi ezinxulumene ne-lipid metabolism kunye ne-steroid hormone biosynthesis. Iziphumo zethu zibonisa ukuba i-PPA inokutshintsha i-gut microbiota kunye neendlela zayo ze-metabolic ezinxulumene nayo. Olu tshintsho lubonweyo lubonisa ukuba izithintelo ezichongiweyo njengezikhuselekileyo ukutyiwa zinokuchaphazela ukwakheka kwe-gut microbiota, kwaye, nempilo yabantu. Phakathi kwazo, i-P, i-G okanye i-S ikhethwa ngokuxhomekeke kwinqanaba lohlulo oluhlalutywayo. Ukunciphisa impembelelo yohlulo olungalunganga, kwamkelwe umda omncinci wobuninzi obuhambelanayo we-1e-4 (1/10,000 reads). Ngaphambi kohlalutyo lwezibalo, ubunini bobuninzi obuhambelanayo obuxelwe nguBracken (fraction_total_reads) buguqulwe kusetyenziswa utshintsho lwe-log-ratio (CLR) oluphakathi (Aitchison, 1982). Indlela ye-CLR ikhethwe ukuguqulwa kwedatha kuba ayiguquguquki kwisikali kwaye yanele kwiiseti zedatha ezingezizo ezimfutshane (Gloor et al., 2017). Uguquko lwe-CLR lusebenzisa i-logarithm yendalo. Idatha yokubala exelwe nguBracken yenziwe yaqheleka kusetyenziswa i-relative log expression (RLE) (Anders kunye noHuber, 2010). Amanani enziwe kusetyenziswa indibaniselwano ye-matplotlib v. 3.7.1, seaborn v. 3.7.2 kunye ne-sequential logarithms (Gloor et al., 2017). 0.12.2 kunye ne-stantanotations v. 0.5.0 (Hunter, 2007; Waskom, 2021; Charlier et al., 2022). Umlinganiselo weBacillus/Bacteroidetes ubalwe kwisampulu nganye kusetyenziswa ukubalwa kweebhaktheriya eziqhelekileyo. Amaxabiso axelwe kwiitheyibhile ajikeleziswa kwiindawo ezi-4 zedesimali. Isalathisi sokwahluka kweSimpson sibalwe kusetyenziswa iskripthi se-alpha_diversity.py esibonelelwe kwiphakheji yeKrakenTools v. 1.2 (Lu et al., 2022). Ingxelo yeBracken ibonelelwe kwiskripthi kwaye isalathisi seSimpson "Si" sibonelelwe kwipharamitha ye--an. Umahluko obalulekileyo ngobuninzi uchazwe njengomahluko ophakathi we-CLR ≥ 1 okanye ≤ -1. Umahluko ophakathi we-CLR we-±1 ubonisa ukunyuka okuphindwe ka-2.7 kubuninzi bohlobo lwesampulu. Uphawu (+/-) lubonisa ukuba i-taxon ininzi na kwisampulu ye-PPA nakwisampulu yokulawula, ngokwahlukeneyo. Ukubaluleka kufunyenwe kusetyenziswa uvavanyo lukaMann-Whitney U (Virtanen et al., 2020). Kusetyenziswe iStatsmodels v. 0.14 (Benjamini and Hochberg, 1995; Seabold and Perktold, 2010), kwaye inkqubo kaBenjamini-Hochberg yasetyenziswa ukulungisa uvavanyo oluninzi. Ixabiso le-p elilungisiweyo ≤ 0.05 lasetyenziswa njengomda wokumisela ukubaluleka kwezibalo.
I-microbiome yomntu idla ngokubizwa ngokuba “lilungu lokugqibela lomzimba” kwaye idlala indima ebalulekileyo kwimpilo yomntu (uBaquero noNombela, 2012). Ngokukodwa, i-microbiome yamathumbu iyaziwa ngempembelelo yayo kwinkqubo yonke kunye nendima kwimisebenzi emininzi ebalulekileyo. Iibhaktheriya ze-commensal zininzi emathunjini, zihlala kwiindawo ezininzi zendalo, zisebenzisa izondlo, kwaye zikhuphisana nezifo ezinokubakho (uJandhyala et al., 2015). Iinxalenye ezahlukeneyo zebhaktheriya ze-gut microbiota ziyakwazi ukuvelisa izondlo ezibalulekileyo ezifana neevithamini kunye nokukhuthaza ukugaywa (uRowland et al., 2018). Ii-metabolites zebhaktheriya nazo zibonakalisiwe ukuba zinefuthe ekuphuhlisweni kwezicubu kwaye ziphucula iindlela ze-metabolic kunye ne-immune (uHeijtz et al., 2011; uYu et al., 2022). Ukwakheka kwe-microbiome yamathumbu omntu kwahlukile kakhulu kwaye kuxhomekeke kwizinto zemfuza kunye nendalo ezifana nokutya, isini, amayeza, kunye nemeko yempilo (uKumbhare et al., 2019).
Ukutya komama yinxalenye ebalulekileyo yophuhliso lwemveku kunye nolweentsana kwaye ngumthombo obonakalayo weekhompawundi ezinokuchaphazela uphuhliso (Bazer et al., 2004; Innis, 2014). Enye yezo khompawundi inomdla yipropionic acid (PPA), imveliso ye-fatty acid efutshane efunyenwe kwi-bacterial fermentation kunye ne-food additive (den Besten et al., 2013). I-PPA ineempawu zokulwa ne-antibacterial kunye ne-antifungal kwaye ke ngoko isetyenziswa njengesigcini sokutya nakwizicelo zoshishino ukuthintela ukukhula kwesikhunta kunye nebhaktheriya (Wemmenhove et al., 2016). I-PPA ineziphumo ezahlukeneyo kwizicubu ezahlukeneyo. Esibindini, i-PPA ineziphumo zokulwa nokuvuvukala ngokuchaphazela ukubonakaliswa kwe-cytokine kwi-macrophages (Kawasoe et al., 2022). Esi siphumo sokulawula sibonwe nakwezinye iiseli zomzimba, nto leyo ekhokelela ekunciphiseni ukuvuvukala (Haase et al., 2021). Nangona kunjalo, isiphumo esichaseneyo sibonwe engqondweni. Izifundo zangaphambili zibonise ukuba ukuvezwa kwe-PPA kubangela ukuziphatha okufana ne-autism kwiimpuku (El-Ansary et al., 2012). Ezinye izifundo zibonise ukuba i-PPA inokubangela i-gliosis kwaye isebenze iindlela ezibangela ukudumba engqondweni (Abdelli et al., 2019). Ngenxa yokuba i-PPA yi-asidi ebuthathaka, inokusasaza nge-epithelium yamathumbu iye egazini kwaye ngaloo ndlela iwele imiqobo ethintelayo kuquka umqobo wegazi-ingqondo kunye ne-placenta (Stinson et al., 2019), igxininisa ukubaluleka kwe-PPA njenge-metabolite elawulayo eveliswa yibhaktheriya. Nangona indima enokubakho ye-PPA njengento ebangela umngcipheko we-autism isaphandwa okwangoku, iziphumo zayo kubantu abane-autism zinokudlula ngaphaya kokukhuthaza ukwahluka kwemithambo-luvo.
Iimpawu zesisu ezifana norhudo kunye nokuqhina ziqhelekile kwizigulana ezineengxaki zophuhliso lwe-neurodevelopmental (Cao et al., 2021). Izifundo zangaphambili zibonise ukuba i-microbiome yezigulana ezine-autism spectrum disorders (ASD) yahlukile kweyabantu abasempilweni, nto leyo ebonisa ukuba kukho i-gut microbiota dysbiosis (Finegold et al., 2010). Ngokufanayo, iimpawu ze-microbiome zezigulana ezinezifo zamathumbu ezivuthayo, ukutyeba kakhulu, isifo se-Alzheimer, njl. nazo zahlukile kwezo zabantu abasempilweni (Turnbaugh et al., 2009; Vogt et al., 2017; Henke et al., 2019). Nangona kunjalo, ukuza kuthi ga ngoku, akukho budlelwane obubangela obukhoyo phakathi kwe-gut microbiome kunye nezifo ze-neurological okanye iimpawu (Yap et al., 2021), nangona iintlobo ezininzi zeebhaktheriya zicingelwa ukuba zidlala indima kwezinye zezi meko zesifo. Umzekelo, i-Akkermansia, iBacteroides, iClostridium, iLactobacillus, iDesulfovibrio kunye nezinye iintlobo zininzi kakhulu kwi-microbiota yezigulane ezine-autism (Tomova et al., 2015; Golubeva et al., 2017; Cristiano et al., 2018; Zurita et al., 2020). Okuphawulekayo kukuba, iintlobo zamalungu ezinye zezi ntlobo zaziwa ngokuba ne-genes ezinxulumene nemveliso ye-PPA (Reichardt et al., 2014; Yun and Lee, 2016; Zhang et al., 2019; Baur and Dürre, 2023). Ngenxa yeempawu ze-PPA zokulwa namagciwane, ukwandisa ubuninzi bayo kunokuba luncedo ekukhuleni kweebhaktheriya ezivelisa i-PPA (Jacobson et al., 2018). Ke ngoko, indawo etyebileyo ye-PFA inokukhokelela kutshintsho kwi-microbiota yamathumbu, kubandakanya iintsholongwane zesisu, ezinokuba zizinto ezinokubangela iimpawu zesisu.
Umbuzo ophambili kuphando lwe-microbiome kukuba ingaba umahluko kwi-microbial composition ungunobangela okanye uphawu lwezifo ezisisiseko. Inyathelo lokuqala ekucaciseni ubudlelwane obunzima phakathi kokutya, i-gut microbiome, kunye nezifo ze-neurological kukuvavanya imiphumo yokutya kwi-microbial composition. Ngenxa yesi sizathu, sisebenzise i-metagenomic sequencing efundwe ixesha elide ukuthelekisa i-gut microbiomes yenzala yeempuku ezondliwa ukutya okutyebileyo kwe-PPA okanye okuphelelwe yi-PPA. Inzala yondliwa ukutya okufanayo noomama bayo. Sicinge ukuba ukutya okutyebileyo kwe-PPA kuya kubangela utshintsho kwi-gut microbial composition kunye neendlela zokusebenza kwe-microbial, ngakumbi ezo zinxulumene ne-PPA metabolism kunye/okanye imveliso ye-PPA.
Olu phononongo lusebenzise iimpuku ze-FVB/N-Tg(GFAP-GFP)14Mes/J transgenic (Jackson Laboratories) ezikhupha iproteni eluhlaza ekhanyayo (GFP) phantsi kolawulo lomxhasi we-GFAP okhethekileyo we-glia ngokulandela izikhokelo zeKomiti yoKhathalelo lweZilwanyana yeYunivesithi yaseCentral Florida (UCF-IACUC) (Inombolo yeMvume yoKusetyenziswa kweZilwanyana: PROTO202000002). Emva kokulumla, iimpuku zahlaliswa zodwa kwiikheyiji ezineempuku ezi-1-5 zesini ngasinye kwikheyiji nganye. Iimpuku zondliwa ngokukhawuleza ngokutya okucociweyo (ukutya okuqhelekileyo okuvulekileyo, amafutha angama-16 kcal%) okanye ukutya okune-sodium propionate (ukutya okuqhelekileyo okuvulekileyo, amafutha angama-16 kcal%, aqulethe i-5,000 ppm sodium propionate). Isixa se-sodium propionate esisetyenzisiweyo sasilingana ne-5,000 mg PFA/kg ubunzima bokutya bubonke. Olu luxinzelelo oluphezulu lwe-PPA oluvunyiweyo ukuba lusetyenziswe njengesigcini-kutya. Ukulungiselela olu phononongo, iimpuku ezizalayo zondliwa zombini ukutya kangangeeveki ezi-4 ngaphambi kokuba zitshate kwaye zaqhubeka ngexesha lokukhulelwa kwedama. Iimpuku ezizalayo [iimpuku ezingama-22, iilawuli ezili-9 (amadoda ama-6, iimazi ezi-3) kunye ne-PPA ezili-13 (amadoda ama-4, iimazi ezi-9)] zalunyulwa zaza zaqhubeka nokutya okufanayo needama kangangeenyanga ezi-5. Iimpuku ezizalayo zaxhelwa xa zineenyanga ezi-5 ubudala kwaye umxholo welindle lazo lamathumbu waqokelelwa kwaye ekuqaleni wagcinwa kwiityhubhu ze-microcentrifuge eziyi-1.5 ml kwi--20°C zaze zadluliselwa kwi-freezer ye--80°C de i-DNA yomzi iphele kwaye kukhutshwe ii-microbial nucleic acids.
I-DNA yoMzimba isusiwe ngokwemigaqo eguquliweyo (Charalampous et al., 2019). Ngamafutshane, umxholo welindle udluliselwe kwi-500 µl InhibitEX (Qiagen, Cat#/ID: 19593) kwaye igcinwe iqandisiwe. Kusetyenzwa ubuninzi be-1-2 fecal pellets ngokukhutshelwa ngakunye. Umxholo welindle emva koko walungiswa ngomatshini kusetyenziswa i-pestle yeplastiki ngaphakathi kwityhubhu ukwenza i-slurry. Faka iisampulu kwi-10,000 RCF imizuzu emi-5 okanye de iisampulu zifakwe i-pellet, uze uphefumlele i-supernatant kwaye uphinde umise i-pellet kwi-250 µl 1 × PBS. Yongeza isisombululo se-250 µl 4.4% saponin (TCI, inombolo yemveliso S0019) kwisampulu njengesepha yokukhulula ii-membranes zeseli ze-eukaryotic. Iisampulu zixutywe kancinci de zibe bushelelezi kwaye zifakwe kubushushu begumbi imizuzu eli-10. Okulandelayo, ukuze kuphazamiseke iiseli ze-eukaryotic, kongezwa amanzi angama-350 μl angena-nuclease kwisampuli, afakwa kwi-incubation imizuzwana engama-30, kwaza kongezwa i-12 μl 5 M NaCl. Emva koko iisampulu zafakwa kwi-centrifuge kwi-6000 RCF imizuzu emi-5. Bilisa i-supernatant kwaye uphinde uyimise i-pellet kwi-100 μl 1X PBS. Ukususa i-DNA yomninimzi, yongeza i-100 μl HL-SAN buffer (12.8568 g NaCl, 4 ml 1M MgCl2, 36 ml amanzi angena-nuclease) kunye ne-10 μl HL-SAN enzyme (ArticZymes P/N 70910-202). Iisampulu zixutywe kakuhle ngokufaka imibhobho zaza zafakwa kwi-incubated kwi-37 °C imizuzu engama-30 kwi-800 rpm kwi-Eppendorf™ ThermoMixer C. Emva kokufunxa, zifakwe kwi-centrifuge kwi-6000 RCF imizuzu emi-3 zaza zahlanjwa kabini nge-800 µl kunye ne-1000 µl 1X PBS. Ekugqibeleni, phinda uxhome i-pellet kwi-100 µl 1X PBS.
I-DNA epheleleyo yebhaktiriya yahlulwa kusetyenziswa iNew England Biolabs Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, Cat# T3010L). Inkqubo yokusebenza eqhelekileyo enikwe le khithi itshintshiwe kancinci. Faka amanzi angenazo i-nuclease kwi-60°C ngaphambi kokuba usebenze ukuze kulungiswe okokugqibela. Yongeza i-10 µl yeProteinase K kunye ne-3 µl yeRNase A kwisampuli nganye. Emva koko yongeza i-100 µl yeCell Lysis Buffer kwaye udibanise kancinci. Emva koko iisampulu zafakwa kwi-Eppendorf™ ThermoMixer C kwi-56°C kunye ne-1400 rpm ubuncinane iyure e-1 ukuya kuthi ga kwiiyure ezi-3. Iisampulu ezifakwe kwi-centrifuge kwi-12,000 RCF imizuzu emi-3 kwaye i-supernatant evela kwisampuli nganye yadluliselwa kwityhubhu eyahlukileyo ye-1.5 mL microcentrifuge equlethe i-400 µL yesisombululo sokubopha. Emva koko iityhubhu zavuthululwa nge-pulse imizuzwana emi-5-10 ngemizuzwana e-1. Dlulisa lonke ulwelo lwesampuli nganye (malunga ne-600-700 µL) kwikhatriji yesihluzo ebekwe kwityhubhu yokuqokelelwa ehambayo. Iityhubhu zafakwa kwi-centrifuge kwi-1,000 RCF imizuzu emi-3 ukuvumela ukubopha kwe-DNA kokuqala kwaye emva koko zafakwa kwi-centrifuge kwi-12,000 RCF umzuzu o-1 ukususa ulwelo oluseleyo. Ikholamu yesampuli yadluliselwa kwityhubhu entsha yokuqokelelwa yaza yahlanjwa kabini. Kwihlambo lokuqala, yongeza i-500 µL ye-wash buffer kwityhubhu nganye. Jikelezisa ityhubhu izihlandlo ezi-3-5 uze u-centrifuge kwi-12,000 RCF umzuzu o-1. Lahla ulwelo kwityhubhu yokuqokelelwa kwaye ubeke i-filter cartridge kwityhubhu efanayo yokuqokelelwa. Kwihlambo lesibini, yongeza i-500 µL ye-wash buffer kwisihluzo ngaphandle kokujika. Iisampulu zafakwa kwi-centrifuge kwi-12,000 RCF umzuzu o-1. Dlulisa isihluzo kwityhubhu ye-LoBind® eyi-1.5 mL uze wongeze i-100 µL yamanzi ashushu angenazo i-nuclease. Izihluzo zifakwe kubushushu begumbi umzuzu omnye zaza zafakwa kwi-centrifuge kwi-12,000 RCF umzuzu omnye. I-DNA e-Eluted igcinwe kwi--80°C.
Uxinaniso lwe-DNA lulinganiswe kusetyenziswa i-Qubit™ 4.0 Fluorometer. I-DNA yalungiswa kusetyenziswa i-Qubit™ 1X dsDNA High Sensitivity Kit (Cat. No. Q33231) ngokwemiyalelo yomenzi. Ukusasazwa kobude be-DNA fragment kulinganiswe kusetyenziswa i-Aglient™ 4150 okanye i-4200 TapeStation. I-DNA yalungiswa kusetyenziswa ii-Agilent™ Genomic DNA Reagents (Cat. No. 5067-5366) kunye ne-Genomic DNA ScreenTape (Cat. No. 5067-5365). Ukulungiswa kwethala leencwadi kwenziwe kusetyenziswa i-Oxford Nanopore Technologies™ (ONT) Rapid PCR Barcoding Kit (SQK-RPB004) ngokwemiyalelo yomenzi. I-DNA yahlelwa kusetyenziswa i-ONT GridION™ Mk1 sequencer ene-Min106D flow cell (R 9.4.1). Useto lokulandelelana yayilulo: ukubiza okuchanekileyo kwesiseko, ixabiso elincinci le-q eliyi-9, ukuseta ibhakhowudi, kunye nokunciphisa ibhakhowudi. Iisampulu zalandelelaniswa iiyure ezingama-72, emva koko idatha yocingo olusisiseko yathunyelwa ukuze iqwalaselwe kwaye ihlalutywe ngakumbi.
Ukucutshungulwa kwe-Bioinformatics kwenziwe kusetyenziswa iindlela ezichazwe ngaphambili (Greenman et al., 2024). Iifayile ze-FASTQ ezifunyenwe ngokulandelana zahlulwe kwiidirectory zesampulu nganye. Ngaphambi kohlalutyo lwe-bioinformatics, idatha yacutshungulwa kusetyenziswa umbhobho olandelayo: okokuqala, iifayile ze-FASTQ zeesampulu zadityaniswa kwifayile enye ye-FASTQ. Emva koko, ukufunda okufutshane kune-1000 bp kwahluzwa kusetyenziswa i-Filtlong v. 0.2.1, kunye neparameter ekuphela kwayo etshintshiweyo yi- –min_length 1000 (Wick, 2024). Ngaphambi kokuhluza ngakumbi, umgangatho wokufunda walawulwa kusetyenziswa i-NanoPlot v. 1.41.3 kunye neeparameter ezilandelayo: –fastq –plots dot –N50 -o
Kwindlela yokwahlulahlula i-taxonomic, ii-reads kunye nee-contigs ezihlanganisiweyo zahlulwa kusetyenziswa iKraken2 v. 2.1.2 (Wood et al., 2019). Yenza iingxelo kunye neefayile zemveliso ze-reads kunye nee-assembly, ngokulandelelana. Sebenzisa ukhetho lwe-–use-names ukuhlalutya ii-reads kunye nee-assembly. Iinketho ze-–gzip-compressed kunye ne-paired zichaziwe kwii-read segments. Ubuninzi be-taxa kwi-metagenomes buqikelelwe kusetyenziswa i-Bracken v. 2.8 (Lu et al., 2017). Siqale sadala i-database ye-kmer equlethe iziseko ezili-1000 sisebenzisa i-bracken-build kunye neeparameter ezilandelayo: -d
Inkcazelo yezakhi zofuzo kunye noqikelelo lobuninzi obuhambelanayo lwenziwe kusetyenziswa inguqulelo eguquliweyo yeprotocol echazwe nguMaranga et al. (Maranga et al., 2023). Okokuqala, ii-contigs ezimfutshane kune-500 bp zisusiwe kuzo zonke ii-assembly kusetyenziswa i-SeqKit v. 2.5.1 (Shen et al., 2016). Ii-assembly ezikhethiweyo emva koko zadityaniswa zaba yi-pan-metagenome. Ii-Open reading frames (ORFs) zachongwa kusetyenziswa i-Prodigal v. 1.0.1 (inguqulelo efanayo ye-Prodigal v. 2.6.3) kunye neeparameter ezilandelayo: -d
Iijini zaqala ukuhlelwa ngokwezihlonzi zeKyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO) ezinikwe yi-eggNOG ukuthelekisa ubukho bendlela yejini. Iijini ezingenayo i-knockouts okanye iijini ezine-knockouts ezininzi zasuswa ngaphambi kohlalutyo. Ubuninzi obuphakathi be-KO nganye kwisampulu nganye babalwe kwaye kwenziwa uhlalutyo lwezibalo. Iijini ze-metabolism ze-PPA zachazwa njengalo naluphi na uhlobo lwejini olunikwe umqolo we-ko00640 kwikholamu ye-KEGG_Pathway, ebonisa indima kwi-metabolism ye-propionate ngokwe-KEGG. Iijini ezichongiweyo njengezinxulumene nemveliso ye-PPA zidweliswe kwiTheyibhile eyoNgezelelweyo 1 (Reichardt et al., 2014; Yang et al., 2017). Uvavanyo lwe-permutation lwenziwa ukuchonga i-metabolism ye-PPA kunye neejini zemveliso ezazininzi kakhulu kuhlobo ngalunye lwesampulu. Ii-permutation eziliwaka zenziwe kwijini nganye ehlalutyiweyo. Ixabiso le-p elingu-0.05 lasetyenziswa njenge-cutoff ukumisela ukubaluleka kwezibalo. Ii-functional annotations zanikwa iijini nganye ngaphakathi kweqela ngokusekelwe kwiinkcazo zeejini ezimeleyo ngaphakathi kweqela. I-Taxa enxulumene ne-metabolism ye-PPA kunye/okanye imveliso ye-PPA inokuchongwa ngokufanisa ii-contig ID kwiifayile zemveliso zeKraken2 kunye nee-contig ID ezifanayo ezigcinwe ngexesha le-functional annotation kusetyenziswa i-eggNOG. Uvavanyo lokubaluleka lwenziwe kusetyenziswa uvavanyo lweMann-Whitney U oluchazwe ngaphambili. Ukulungiswa kovavanyo oluninzi kwenziwe kusetyenziswa inkqubo yeBenjamini-Hochberg. Ixabiso le-p le-≤ 0.05 lisetyenzisiwe njenge-cutoff ukumisela ukubaluleka kwezibalo.
Ukwahluka kwe-microbiome yamathumbu eempuku kuhlolwe kusetyenziswa i-Simpson diversity index. Akukho mahluko abalulekileyo abonwe phakathi kweesampulu zolawulo kunye ne-PPA ngokwe-genus kunye ne-species diversity (ixabiso le-p kwi-genus: 0.18, ixabiso le-p kwi-species: 0.16) (Umfanekiso 1). Ukwakheka kwe-microbial emva koko kwathelekiswa kusetyenziswa uhlalutyo lwe-principal component (PCA). Umfanekiso 2 ubonisa ukuhlanganiswa kweesampulu nge-phyla yazo, nto leyo ebonisa ukuba kukho umahluko kwi-species ye-microbiomes phakathi kwe-PPA kunye neesampulu zolawulo. Olu kuhlanganiswa aluzange lubonakale kakhulu kwinqanaba le-genus, nto leyo ebonisa ukuba i-PPA ichaphazela iibhaktheriya ezithile (Umfanekiso Ongezelelweyo 1).
Umfanekiso 1. Ukwahluka kwe-alpha kwe-genera kunye nokwakheka kweentlobo ze-mouse gut microbiome. Iiploti zebhokisi ezibonisa i-Simpson diversity indices ze-genera (A) kunye ne-species (B) kwi-PPA kunye neesampulu zolawulo. Ukubaluleka kuchongiwe kusetyenziswa uvavanyo lweMann-Whitney U, kwaye ukulungiswa okuninzi kwenziwe kusetyenziswa inkqubo yeBenjamini-Hochberg. ns, ixabiso le-p lalingabalulekanga (p>0.05).
Umfanekiso 2. Iziphumo zohlalutyo lwecandelo eliphambili lolwakhiwo lwe-microbiome yamathumbu empuku kwinqanaba leentlobo. Iploti yohlalutyo lwecandelo eliphambili ibonisa ukusasazwa kweesampuli kuzo zombini iinxalenye zazo eziphambili. Imibala ibonisa uhlobo lwesampulu: Iimpuku ezivezwe yi-PPA zimfusa kwaye iimpuku zokulawula zimthubi. Iinxalenye eziphambili 1 kunye no-2 zidwetshwe kwi-x-axis kunye ne-y-axis, ngokwahlukeneyo, kwaye zibonakaliswa njengomlinganiselo wazo wokuguquguquka ochaziweyo.
Ukusebenzisa idatha yokubala eguquliweyo ye-RLE, ukwehla okukhulu kwi-median Bacteroidetes/Bacilli ratio kubonwe kwi-control kunye neempuku ze-PPA (control: 9.66, PPA: 3.02; p-value = 0.0011). Lo mahluko ubangelwe kubuninzi obuphezulu beeBacteroidetes kwiimpuku ze-PPA xa kuthelekiswa nezolawulo, nangona umahluko wawungabalulekanga (control mean CLR: 5.51, PPA mean CLR: 6.62; p-value = 0.054), ngelixa ubuninzi beeBacteroidetes bufana (control mean CLR: 7.76, PPA mean CLR: 7.60; p-value = 0.18).
Uhlalutyo lobuninzi bamalungu e-taxonomic kwi-gut microbiome lubonise ukuba i-phylum e-1 kunye neentlobo ezingama-77 zahluke kakhulu phakathi kwe-PPA kunye neesampuli zolawulo (Itheyibhile eyongezelelweyo 2). Ubuninzi beentlobo ezingama-59 kwiisampulu ze-PPA babuphezulu kakhulu kunobo kwiisampuli zolawulo, ngelixa ubuninzi beentlobo ezili-16 kuphela kwiisampulu zolawulo buphezulu kunobo kwiisampuli ze-PPA (Umfanekiso 3).
Umfanekiso 3. Ubuninzi obahlukeneyo be-taxa kwi-microbiome yamathumbu e-PPA kunye neempuku zokulawula. Iiploti zevolcano zibonisa umahluko kubuninzi be-genera (A) okanye iintlobo (B) phakathi kwe-PPA kunye neesampuli zokulawula. Amachaphaza angwevu abonisi mahluko mkhulu kubuninzi be-taxa. Amachaphaza anemibala abonisa umahluko obalulekileyo kubuninzi (ixabiso le-p ≤ 0.05). Amachaphaza aphezulu angama-20 anomahluko mkhulu kubuninzi phakathi kweentlobo zeesampuli aboniswe kwi-red and light blue (control and PPA samples), ngokwahlukeneyo. Amachaphaza atyheli namfusa ayeninzi ubuncinane ngokuphindwe ka-2.7 kwiisampulu zokulawula okanye ze-PPA kunakwiisampulu zokulawula. Amachaphaza amnyama amele i-taxa enobuninzi obahlukeneyo kakhulu, kunye nomahluko ophakathi we-CLR phakathi kwe--1 kunye ne-1. Amaxabiso e-P abalwe kusetyenziswa uvavanyo lweMann-Whitney U kwaye alungiswa kuvavanyo oluninzi kusetyenziswa inkqubo yeBenjamini-Hochberg. Umahluko ophakathi kwe-CLR ocacileyo ubonisa umahluko obalulekileyo kubuninzi.
Emva kokuhlalutya ukwakheka kwe-microbial emathunjini, senze isishwankathelo esisebenzayo se-microbiome. Emva kokucoca ii-genes ezikumgangatho ophantsi, kwachongwa ii-genes ezizodwa ezingama-378,355 kuzo zonke iisampuli. Ubuninzi obuguquliweyo bezi genes busetyenziselwe uhlalutyo lwe-principal component (PCA), kwaye iziphumo zibonise izinga eliphezulu lokuhlanganiswa kweentlobo zeesampuli ngokusekelwe kwiiprofayili zazo zokusebenza (Umfanekiso 4).
Umfanekiso 4. Iziphumo ze-PCA zisebenzisa iprofayili esebenzayo ye-mouse gut microbiome. I-PCA plot ibonisa ukusasazwa kweesampuli kuzo zonke izinto zazo zokuqala ezimbini eziphambili. Imibala ibonisa uhlobo lwesampulu: Iimpuku ezivezwe yi-PPA zimfusa kwaye iimpuku zokulawula zimthubi. Izinto eziphambili 1 kunye no-2 zidwetshwe kwi-x-axis kunye ne-y-axis, ngokwahlukeneyo, kwaye zibonakaliswa njengomlinganiselo wazo wokuguquguquka ochaziweyo.
Emva koko sihlolisise ubuninzi bee-knockouts ze-KEGG kwiintlobo ezahlukeneyo zeesampuli. Kuchongwe ii-knockouts ezizodwa ezingama-3648, apho ezili-196 zazininzi kakhulu kwiisampuli zolawulo kwaye ezili-106 zazininzi ngakumbi kwiisampuli ze-PPA (Umfanekiso 5). Iijini ezili-145 zifunyenwe kwiisampuli zolawulo kunye neejini ezingama-61 kwiisampuli ze-PPA, ezinobuninzi obahlukeneyo kakhulu. Iindlela ezinxulumene ne-lipid kunye ne-aminosugar metabolism zityebile kakhulu kwiisampuli ze-PPA (Itheyibhile eyongezelelweyo 3). Iindlela ezinxulumene ne-nitrogen metabolism kunye neenkqubo ze-sulfur relay zityebile kakhulu kwiisampuli zolawulo (Itheyibhile eyongezelelweyo 3). Ubuninzi beejini ezinxulumene ne-aminosugar/nucleotide metabolism (ko:K21279) kunye ne-inositol phosphate metabolism (ko:K07291) ziphezulu kakhulu kwiisampuli ze-PPA (Umfanekiso 5). Iisampuli zolawulo zazineejini ezininzi kakhulu ezinxulumene ne-benzoate metabolism (ko:K22270), i-nitrogen metabolism (ko:K00368), kunye ne-glycolysis/gluconeogenesis (ko:K00131) (Umfanekiso 5).
Umzobo 5. Ubuninzi obahlukeneyo bee-KO kwi-microbiome yamathumbu e-PPA kunye neempuku zokulawula. Iploti yentaba-mlilo ibonisa umahluko kubuninzi bamaqela asebenzayo (ii-KO). Amachaphaza angwevu abonisa ii-KO ezinobuninzi obungahlukanga kakhulu phakathi kweentlobo zeesampuli (ixabiso le-p > 0.05). Amachaphaza anemibala abonisa umahluko obalulekileyo kubuninzi (ixabiso le-p ≤ 0.05). Ii-KO ezingama-20 ezinomahluko omkhulu kubuninzi phakathi kweentlobo zeesampuli ziboniswe ngombala obomvu nokhanyayo oluhlaza okwesibhakabhaka, ohambelana neesampuli zolawulo kunye ne-PPA, ngokwahlukeneyo. Amachaphaza atyheli namfusa abonisa ii-KO ezazininzi ngokuphindwe kayi-2.7 kulawulo kunye neesampuli ze-PPA, ngokwahlukeneyo. Amachaphaza amnyama abonisa ii-KO ezinobuninzi obahlukeneyo kakhulu, kunye nomahluko ophakathi we-CLR phakathi kwe--1 kunye ne-1. Amaxabiso e-P abalwe kusetyenziswa uvavanyo lweMann-Whitney U kwaye ahlengahlengiswa ukuthelekisa okuninzi kusetyenziswa inkqubo yeBenjamini-Hochberg. I-NaN ibonisa ukuba i-KO ayiyoyendlela kwi-KEGG. Amaxabiso omahluko phakathi kwe-CLR angqindilili abonisa umahluko obalulekileyo kubuninzi. Ukuze ufumane ulwazi oluthe kratya malunga neendlela eziya kuzo ezi KO zidwelisiweyo, jonga iTheyibhile eyoNgezelelweyo 3.
Phakathi kwezakhi zofuzo ezichaziweyo, ii-genes ezili-1601 zazinobuninzi obahlukeneyo phakathi kweentlobo zeesampuli (p ≤ 0.05), apho i-gene nganye yayininzi ngokuphindwe kayi-2.7. Kwezi zakhi zofuzo, ii-genes ezi-4 zazininzi kakhulu kwiisampulu zolawulo kwaye ii-genes ezili-1597 zazininzi kakhulu kwiisampulu ze-PPA. Ngenxa yokuba i-PPA ineempawu zokulwa neentsholongwane, sihlolisise ubuninzi be-metabolism ye-PPA kunye nee-genes zokuvelisa phakathi kweentlobo zeesampuli. Phakathi kwezakhi zofuzo ezinxulumene ne-metabolism ye-PPA eziyi-1332, ii-genes ezingama-27 zazininzi kakhulu kwiisampulu zolawulo kwaye ii-genes ezili-12 zazininzi ngakumbi kwiisampulu ze-PPA. Phakathi kwezakhi zofuzo ezinxulumene nemveliso ye-PPA ezingama-223, i-gene e-1 yayininzi kakhulu kwiisampulu ze-PPA. Umfanekiso 6A ubonisa ngakumbi ubuninzi obukhulu bezakhi zofuzo ezibandakanyeka kwi-metabolism ye-PPA, ngobuninzi obukhulu kakhulu kwiisampulu zolawulo kunye nobukhulu obukhulu bempembelelo, ngelixa Umfanekiso 6B ugxininisa ii-genes zomntu ngamnye ezinobuninzi obukhulu kakhulu obubonwe kwiisampulu ze-PPA.
Umzobo 6. Ubuninzi obahlukeneyo be-genes ezinxulumene ne-PPA kwi-mouse gut microbiome. Iiploti zevolcano zibonisa umahluko kubuninzi be-genes ezinxulumene ne-PPA metabolism (A) kunye nemveliso ye-PPA (B). Amachaphaza angwevu abonisa ii-genes ezinobuninzi obungahlukanga kakhulu phakathi kweentlobo zeesampulu (ixabiso le-p > 0.05). Amachaphaza anemibala abonisa umahluko obalulekileyo kubuninzi (ixabiso le-p ≤ 0.05). Ii-genes ezingama-20 ezinomahluko omkhulu kubuninzi ziboniswe kwi-red and light blue (control and PPA samples), ngokwahlukeneyo. Ubuninzi be-yellow dots and purple babungaphezulu ngokuphindwe kayi-2.7 kwi-control and PPA samples kunakwi-control samples. Amachaphaza amnyama amele ii-genes ezinobuninzi obahlukeneyo kakhulu, kunye nomahluko we-CLR ophakathi phakathi kwe--1 kunye ne-1. Amaxabiso e-P abalwe kusetyenziswa uvavanyo lweMann-Whitney U kwaye alungiswa ukuthelekisa okuninzi kusetyenziswa inkqubo yeBenjamini-Hochberg. Ii-genes zihambelana nee-genes ezimeleyo kwikhathalogu ye-gene engeyiyo i-redundant. Amagama e-gene aqulathe uphawu lwe-KEGG oluchaza i-KO gene. Umahluko we-CLR ongqindilili ophakathi ubonisa ubuninzi obahlukeneyo kakhulu. Ideshi (-) ibonisa ukuba akukho simboli yejini kwisiseko sedatha se-KEGG.
I-Taxa enezakhi zofuzo ezinxulumene ne-metabolism ye-PPA kunye/okanye imveliso zichongiwe ngokufanisa ubume be-taxonomic be-contigs kunye ne-contig ID yezakhi zofuzo. Kwinqanaba le-genus, ii-genera ezili-130 zifunyenwe zinezakhi zofuzo ezinxulumene ne-metabolism ye-PPA kwaye ii-genera ezingama-61 zifunyenwe zinezakhi zofuzo ezinxulumene nemveliso ye-PPA (Itheyibhile eyongezelelweyo 4). Nangona kunjalo, akukho genera ibonise umahluko omkhulu ngobuninzi (p > 0.05).
Kwinqanaba leentlobo, iintlobo zebhaktiriya ezili-144 zifunyenwe zinezakhi zofuzo ezinxulumene ne-metabolism ye-PPA kwaye iintlobo zebhaktiriya ezingama-68 zifunyenwe zinezakhi zofuzo ezinxulumene nemveliso ye-PPA (Itheyibhile Eyongezelelweyo 5). Phakathi kwe-metabolism ye-PPA, iibhaktiriya ezisibhozo zibonise ukwanda okukhulu kobuninzi phakathi kweentlobo zeesampuli, kwaye zonke zibonise utshintsho olukhulu ekusebenzeni (Itheyibhile Eyongezelelweyo 6). Zonke ii-metabolism ze-PPA ezichongiweyo ezinomahluko omkhulu ngobuninzi bezininzi ngakumbi kwiisampuli ze-PPA. Ukuhlelwa kwinqanaba leentlobo kubonise abameli bezizukulwana abangafaniyo kakhulu phakathi kweentlobo zeesampuli, kubandakanya iintlobo ezininzi zeBacteroides kunye neRuminococcus, kunye neDuncania dubois, Myxobacterium enterica, Monococcus pectinolyticus, kunye neAlcaligenes polymorpha. Phakathi kweebhaktiriya ezivelisa i-PPA, iibhaktiriya ezine zibonise umahluko omkhulu ngobuninzi phakathi kweentlobo zeesampuli. Iintlobo ezinomahluko omkhulu ngobuninzi ziquka iBacteroides novorossi, Duncania dubois, Myxobacterium enteritidis, kunye neRuminococcus bovis.
Kolu phononongo, sihlolisise imiphumo yokuvezwa yi-PPA kwi-gut microbiota yeempuku. I-PPA inokubangela iimpendulo ezahlukeneyo kwiibhaktheriya kuba iveliswa ziintlobo ezithile, isetyenziswa njengomthombo wokutya zezinye iintlobo, okanye ineempembelelo zokulwa nentsholongwane. Ke ngoko, ukongezwa kwayo kwindawo yamathumbu ngokutya okunesondlo kunokuba nemiphumo eyahlukileyo kuxhomekeke ekunyamezeleni, ekuthambekeni, kunye nokukwazi ukuyisebenzisa njengomthombo wezondlo. Iintlobo zebhaktheriya ezinobuthathaka zinokususwa kwaye zitshintshwe zezo zixhathisa ngakumbi kwi-PPA okanye ezikwaziyo ukuyisebenzisa njengomthombo wokutya, nto leyo ekhokelela kutshintsho ekubunjweni kwe-gut microbiota. Iziphumo zethu zityhile umahluko omkhulu ekubunjweni kweentsholongwane kodwa akukho mpembelelo kulwahluko lweentsholongwane ngokubanzi. Imiphumo emikhulu ibonwe kwinqanaba leentlobo, kunye nee-taxa ezingaphezu kwama-70 ezahlukileyo kakhulu phakathi kwe-PPA kunye neesampuli zolawulo (Itheyibhile eyongezelelweyo 2). Uvavanyo olongezelelweyo lokwakheka kweesampuli ezivezwe yi-PPA lubonise ukungafani okukhulu kweentlobo zentsholongwane xa kuthelekiswa neesampuli ezingavezwanga, nto leyo ebonisa ukuba i-PPA inokuphucula iimpawu zokukhula kweentsholongwane kwaye inciphise inani leentsholongwane ezinokusinda kwiindawo ezinobutyebi be-PPA. Ke ngoko, i-PPA inokubangela utshintsho ngokukhetha kunokuba ibangele ukuphazamiseka okubanzi kolwahluko lweentsholongwane ze-gut microbiota.
Izithinteli zokutya ezifana ne-PPA ngaphambili zibonakalisiwe ukuba zitshintsha ubuninzi bezinto ezibangela iintsholongwane emathunjini ngaphandle kokuchaphazela ukwahlukana ngokubanzi (Nagpal et al., 2021). Apha, sibone umahluko omkhulu phakathi kweentlobo zeBacteroidetes ngaphakathi kwi-phylum Bacteroidetes (ezazisaziwa ngokuba ziiBacteroidetes), ezazityebiswe kakhulu kwiimpuku ezivezwe yi-PPA. Ukwanda kobuninzi beentlobo zeBacteroides kunxulunyaniswa nokwanda kokonakala kwe-mucus, okunokunyusa umngcipheko wosulelo kunye nokukhuthaza ukudumba (Cornick et al., 2015; Desai et al., 2016; Penzol et al., 2019). Olunye uphando lufumanise ukuba iimpuku zamadoda ezisandula ukuzalwa ezinyangwe ngeBacteroides fragilis zibonakalise ukuziphatha kwezentlalo okukhumbuza i-autism spectrum disorder (ASD) (Carmel et al., 2023), kwaye ezinye izifundo zibonise ukuba iintlobo zeBacteroides zinokutshintsha umsebenzi wokuzikhusela komzimba kwaye zikhokelele kwi-cardiomyopathy yokuvuvukala komzimba (Gil-Cruz et al., 2019). Iintlobo zesizukulwana iRuminococcus, iPrevotella, kunye neParabacteroides nazo zanda kakhulu kwiimpuku ezivezwe kwiPPA (Coretti et al., 2018). Ezinye iintlobo zeRuminococcus zidibene nezifo ezifana nesifo sikaCrohn ngokuveliswa kwee-cytokines ezibangela ukuvuvukala (Henke et al., 2019), ngelixa iintlobo zePrevotella ezifana nePrevotella humani zidibene nezifo zemetabolism ezifana noxinzelelo lwegazi kunye nokuziva i-insulin (Pedersen et al., 2016; Li et al., 2017). Ekugqibeleni, sifumanise ukuba umlinganiselo weBacteroidetes (ezazisaziwa ngaphambili njengeFirmicutes) kwiBacteroidetes wawuphantsi kakhulu kwiimpuku ezivezwe yiPPA kunakwiimpuku ezilawulayo ngenxa yobuninzi obukhulu beentlobo zeBacteroidetes. Olu linganiselo ngaphambili luboniswe njengophawu olubalulekileyo lwe-homeostasis yamathumbu, kwaye ukuphazamiseka kolu linganiselo kunxulunyaniswe neemeko ezahlukeneyo zezifo (Turpin et al., 2016; Takezawa et al., 2021; An et al., 2023), kubandakanya izifo zamathumbu ezivuthayo (Stojanov et al., 2020). Ngokudibeneyo, iintlobo ze-phylum Bacteroidetes zibonakala zichaphazeleka kakhulu yi-PPA ephezulu yokutya. Oku kusenokubangelwa kukunyamezela okuphezulu kwi-PPA okanye ukukwazi ukusebenzisa i-PPA njengomthombo wamandla, okuye kwabonakala kuyinyani ubuncinane kuhlobo olunye, uHoylesella enocea (Hitch et al., 2022). Ngaphandle koko, ukuvezwa kwe-PPA yomama kunokuphucula uphuhliso losana ngokwenza amathumbu eempuku abe sesichengeni sokuhlaselwa yi-Bacteroidetes; nangona kunjalo, uyilo lwethu lophando aluzange luvumele uvavanyo olunjalo.
Uvavanyo lomxholo we-metagenomic lubonise umahluko omkhulu kubuninzi be-genes ezinxulumene ne-metabolism ye-PPA kunye nemveliso, apho iimpuku ezivezwe yi-PPA zibonisa ubuninzi be-genes ezinoxanduva lokuveliswa kwe-PPA, ngelixa iimpuku ezingengo-PPA-exposed zibonise ubuninzi be-genes ezinoxanduva lokusetyenziswa kwe-PAA (Umfanekiso 6). Ezi ziphumo zibonisa ukuba isiphumo se-PPA kwi-microbial composition asinakuba ngenxa yokusetyenziswa kwayo kuphela, kungenjalo ubuninzi be-genes ezinxulumene ne-PPA metabolism bekufanele ukuba bubonise ubuninzi obukhulu kwi-microbiome yamathumbu eempuku ezivezwe yi-PPA. Enye ingcaciso kukuba i-PPA ilawula ubuninzi bebhaktheriya ngokuyintloko ngemiphumo yayo yokulwa neentsholongwane endaweni yokusetyenziswa kwayo yiibhaktheriya njengesondlo. Izifundo zangaphambili zibonise ukuba i-PPA ithintela ukukhula kweSalmonella Typhimurium ngendlela exhomekeke kwidosi (Jacobson et al., 2018). Ukuvezwa kumanqanaba aphezulu e-PPA kunokukhetha iibhaktheriya ezichasene neempawu zayo zokulwa neentsholongwane kwaye zisenokungakwazi ukuyigaya okanye ukuyivelisa. Umzekelo, iintlobo ezininzi zeParabacteroides zibonise ubuninzi obuphezulu kakhulu kwiisampuli zePPA, kodwa akukho genes ezinxulumene ne-metabolism yePPA okanye imveliso ezifunyenweyo (Iitheyibhile ezongezelelweyo 2, 4, kunye no-5). Ngaphezu koko, imveliso yePPA njengemveliso yokubilisa isasazwa kakhulu kwiibhaktheriya ezahlukeneyo (Gonzalez-Garcia et al., 2017). Ukwahluka okuphezulu kweebhaktheriya kunokuba sisizathu sobuninzi obukhulu beejini ezinxulumene ne-metabolism yePPA kwiisampuli zolawulo (Averina et al., 2020). Ngaphezu koko, zi-27 kuphela (2.14%) zeejini ezili-1332 ezaxelwa kwangaphambili ukuba ziijini ezinxulumene ne-metabolism yePPA kuphela. Iijini ezininzi ezinxulumene ne-metabolism yePPA nazo zibandakanyeka kwezinye iindlela ze-metabolism. Oku kubonisa ngakumbi ukuba ubuninzi beejini ezibandakanyeka kwi-metabolism yePPA buphezulu kwiisampuli zolawulo; ezi jini zinokusebenza kwiindlela ezingabangela ukusetyenziswa okanye ukwakheka kwePPA njengemveliso engasemva. Kule meko, yijini enye kuphela enxulumene ne-PPA evelisiweyo ibonise umahluko omkhulu kubuninzi phakathi kweentlobo zeesampuli. Ngokungafaniyo neejini ezinxulumene ne-metabolism yePPA, iijini eziphawulayo zemveliso yePPA zikhethwe kuba zibandakanyeka ngokuthe ngqo kwindlela yebhaktheriya yemveliso yePPA. Kwiimpuku ezivezwe yi-PPA, zonke iintlobo zezilwanyana zifunyenwe zinobuninzi obuninzi kunye namandla okuvelisa i-PPA. Oku kuxhasa ukuqikelela ukuba ii-PPA ziya kukhetha abavelisi be-PPA kwaye ke ngoko ziqikelela ukuba amandla okuvelisa i-PPA aya kwanda. Nangona kunjalo, ubuninzi be-gene abuhambelani nokubonakaliswa kwe-gene; ngoko ke, nangona ubuninzi be-genes ezinxulumene ne-metabolism ye-PPA buphezulu kwiisampulu zolawulo, izinga lokubonakaliswa linokwahluka (Shi et al., 2014). Ukuqinisekisa ubudlelwane phakathi kokuxhaphaka kwe-genes ezivelisa i-PPA kunye nemveliso ye-PPA, kufuneka izifundo zokubonakaliswa kwe-genes ezibandakanyekayo kwimveliso ye-PPA.
Isichazi-magama esisebenzayo se-PPA kunye nee-metagenomes zolawulo sityhile umahluko othile. Uhlalutyo lwe-PCA lomxholo we-gene lubonise amaqela ahlukeneyo phakathi kwe-PPA kunye neesampuli zolawulo (Umfanekiso 5). Ukuhlanganiswa kwesampulu ngaphakathi kutyhile ukuba umxholo we-gene yolawulo wawuhluke kakhulu, ngelixa iisampuli ze-PPA ziqokelelene ndawonye. Ukuhlanganiswa ngokomxholo we-gene kwakufana nokuhlanganiswa ngokweentlobo. Ke ngoko, umahluko kubuninzi bendlela uhambelana notshintsho kubuninzi beentlobo ezithile kunye neentlobo ngaphakathi kwazo. Kwiisampuli ze-PPA, iindlela ezimbini ezinobuninzi obukhulu kakhulu zazinxulumene ne-aminosugar/nucleotide sugar metabolism (ko:K21279) kunye neendlela ezininzi ze-lipid metabolism (ko:K00647, ko:K03801; Itheyibhile eyongeziweyo 3). Iijini ezinxulumene ne-ko:K21279 zaziwa ukuba zinxulumene ne-genus Bacteroides, enye ye-genera enenani eliphezulu kakhulu leentlobo kwiisampuli ze-PPA. Le enzyme inokuphepha impendulo yomzimba ngokuveza ii-capsular polysaccharides (Wang et al., 2008). Oku kunokubangela ukwanda kwee-Bacteroidetes ezibonwe kwiimpuku ezivezwe yi-PPA. Oku kuhambelana nokwanda kokwenziwa kwe-fatty acid ebonwe kwi-PPA microbiome. Iibhaktheriya zisebenzisa indlela ye-FASIIko:K00647 (fabB) ukuvelisa ii-fatty acids, ezinokuchaphazela iindlela ze-metabolic zomntu ohlala kuyo (Yao and Rock, 2015; Johnson et al., 2020), kwaye utshintsho kwi-lipid metabolism lunokudlala indima ekuphuhlisweni kwe-neurodevelopment (Yu et al., 2020). Enye indlela ebonisa ukwanda kobuninzi kwiisampulu ze-PPA yayiyi-steroid hormone biosynthesis (ko:K12343). Kukho ubungqina obukhulayo bokuba kukho ubudlelwane obuchaseneyo phakathi kwamandla e-gut microbiota okuchaphazela amanqanaba e-hormone kunye nokuchaphazeleka zii-hormone, kangangokuba amanqanaba aphezulu e-steroid anokuba nemiphumo emibi kwimpilo (Tetel et al., 2018).
Olu phononongo alunazo izithintelo kunye nokuqwalaselwa. Umahluko obalulekileyo kukuba asizange senze uvavanyo lwe-physiological lwezilwanyana. Ke ngoko, akunakwenzeka ukugqiba ngokuthe ngqo ukuba utshintsho kwi-microbiome ludibene nasiphi na isifo. Enye into ekufuneka iqwalaselwe kukuba iimpuku kolu phononongo zondliwa ukutya okufanayo noomama bazo. Izifundo zexesha elizayo zinokugqiba ukuba ukutshintsha ukusuka ekutyeni okutyebileyo kwi-PPA ukuya ekutyeni okungena-PPA kuyayiphucula na imiphumo yako kwi-microbiome. Omnye umda wolu phononongo lwethu, njengabanye abaninzi, bubungakanani besampulu obulinganiselweyo. Nangona izigqibo ezifanelekileyo zinokuthathwa, ubungakanani besampulu obukhulu buya kubonelela ngamandla amakhulu ezibalo xa kuhlalutywa iziphumo. Sikwalumkile malunga nokufikelela kwizigqibo malunga nonxulumano phakathi kotshintsho kwi-microbiome yamathumbu kunye nasiphi na isifo (Yap et al., 2021). Izinto ezididayo kuquka ubudala, isini, kunye nokutya zinokuchaphazela kakhulu ukwakheka kwe-microorganisms. Ezi zinto zinokuchaza ukungangqinelani okubonwe kwiincwadi malunga nonxulumano lwe-microbiome yamathumbu nezifo ezintsonkothileyo (Johnson et al., 2019; Lagod and Naser, 2023). Umzekelo, amalungu e-genus Bacteroidetes abonakalisiwe ukuba ayanda okanye anciphile kwizilwanyana nakubantu abane-ASD (Angelis et al., 2013; Kushak et al., 2017). Ngokufanayo, izifundo zokwakheka kwamathumbu kwizigulane ezinezifo zamathumbu ezivuthayo zifumanise ukwanda kunye nokuncipha kwi-taxa efanayo (Walters et al., 2014; Forbes et al., 2018; Upadhyay et al., 2023). Ukunciphisa impembelelo yocalucalulo ngokwesini, sizamile ukuqinisekisa ukumelwa ngokulinganayo kwezesondo ukuze umahluko ube ngokugqithisileyo uqhutywa kukutya. Omnye umngeni we-functional annotation kukususwa kwe-redundant gene sequences. Indlela yethu ye-gene clustering ifuna i-95% sequence identity kunye ne-85% definition length, kunye ne-90% alignment coverage ukuphelisa i-false clustering. Nangona kunjalo, kwezinye iimeko, sibone ii-COGs ezine-annotations ezifanayo (umz., MUT) (Umzobo 6). Izifundo ezongezelelweyo ziyafuneka ukuze kuchongwe ukuba ezi orthologs zahlukile na, zidibene ne-genera ethile, okanye ukuba oku kungumda wendlela yokuhlanganisa iijini. Omnye umda we-functional annotation kukungahlelwa kakuhle; i-bacterial gene mmdA yi-enzyme eyaziwayo ebandakanyekayo kwi-propionate synthesis, kodwa i-KEGG ayinxulumanisi nendlela ye-propionate metabolic. Ngokwahlukileyo koko, ii-scpB kunye nee-mmcD orthologs zihlobene. Inani elikhulu leejini ezingenazo ii-knockouts ezikhethiweyo zinokubangela ukungakwazi ukuchonga iijini ezinxulumene ne-PPA xa kuvavanywa ubuninzi bejini. Izifundo zexesha elizayo ziya kuzuza kuhlalutyo lwe-metatranscriptome, olunokubonelela ngokuqonda okunzulu kweempawu zokusebenza kwe-gut microbiota kwaye ludibanise ukubonakaliswa kwejini kwimiphumo enokubakho. Kwiizifundo ezibandakanya iingxaki ezithile zophuhliso lwe-neurodevelopmental okanye izifo zamathumbu ezivuthayo, uvavanyo lwe-physiological kunye nokuziphatha kwezilwanyana luyafuneka ukuze kuqhagamshelwe utshintsho kwi-microbiome composition kwezi ngxaki. Izifundo ezongezelelweyo zokutshintsha i-gut microbiome kwiimpuku ezingenazo iintsholongwane nazo ziya kuba luncedo ekuchongeni ukuba i-microbiome ngumqhubi okanye uphawu lwesifo.
Ngamafutshane, sibonise ukuba i-PPA yokutya isebenza njengento ebalulekileyo ekutshintsheni ukwakheka kwe-microbiota yamathumbu. I-PPA sisithinteli esivunyiweyo yi-FDA esifumaneka kakhulu kukutya okwahlukeneyo, okuthi xa sichaphazeleka ixesha elide, kunokubangela ukuphazamiseka kwezityalo eziqhelekileyo zamathumbu. Sifumene utshintsho kubuninzi beebhaktheriya ezininzi, nto leyo ebonisa ukuba i-PPA inokuphembelela ukwakheka kwe-microbiota yamathumbu. Utshintsho kwi-microbiota lunokubangela utshintsho kumanqanaba eendlela ezithile ze-metabolic, nto leyo enokubangela utshintsho lwe-physiological oluhambelana nempilo yomntu. Kufuneka izifundo ezongezelelweyo ukuze kuqinisekiswe ukuba imiphumo ye-PPA yokutya kukwakheka kweentsholongwane inokukhokelela kwi-dysbiosis okanye kwezinye izifo. Olu phononongo lubeka isiseko sezifundo ezizayo malunga nendlela imiphumo ye-PPA kukwakheka kwamathumbu enokuthi ichaphazele ngayo impilo yabantu.
Iiseti zedatha ezivezwe kolu phononongo ziyafumaneka kwiindawo zokugcina ulwazi ezikwi-intanethi. Igama lendawo yokugcina ulwazi kunye nenombolo yokungena kwezi: https://www.ncbi.nlm.nih.gov/, PRJNA1092431.
Olu phononongo lwezilwanyana luvunyiwe yiKomiti yoLondolozo lweZilwanyana yeYunivesithi yaseCentral Florida (UCF-IACUC) (Inombolo yeMvume yokusetyenziswa kweZilwanyana: PROTO202000002). Olu phononongo luhambelana nemithetho yasekuhlaleni, imigaqo, kunye neemfuno zesikhungo.
NG: Ukuqwalasela, Ukulawula idatha, Uhlalutyo olusesikweni, Uphando, Indlela yokusebenza, Isoftware, Ukubona ngeliso elibukhali, Ukubhala (uyilo lokuqala), Ukubhala (uphononongo kunye nokuhlela). LA: Ukuqwalasela, Ukulawula idatha, Indlela yokusebenza, Izixhobo, Ukubhala (uphononongo kunye nokuhlela). SH: Uhlalutyo olusesikweni, Isoftware, Ukubhala (uphononongo kunye nokuhlela). SA: Uphando, Ukubhala (uphononongo kunye nokuhlela). IJaji eliyiNtloko: Uphando, Ukubhala (uphononongo kunye nokuhlela). SN: Ukuqwalasela, Ulawulo lweeprojekthi, Izixhobo, Ukujonga, Ukubhala (uphononongo kunye nokuhlela). TA: Ukuqwalasela, Ulawulo lweeprojekthi, Ukujonga, Ukubhala (uphononongo kunye nokuhlela).
Ababhali bavakalise ukuba abafumananga nkxaso-mali yophando, ukubhalwa, kunye/okanye ukupapashwa kweli nqaku.
Ababhali bathi olu phando lwenziwe ngaphandle kobudlelwane bezorhwebo okanye bezemali obunokuthathwa njengongquzulwano lwemidla olunokwenzeka. alusebenzi.
Zonke izimvo ezivezwe kweli nqaku zezababhali kuphela kwaye azibonisi ngokuyimfuneko izimvo zamaziko abo, abapapashi, abahleli, okanye abahlaziyi. Naziphi na iimveliso ezivavanyiweyo kweli nqaku, okanye naziphi na izibango ezenziwe ngabavelisi bazo, aziqinisekiswanga okanye azivunywa ngumshicileli.
Izinto ezongezelelweyo zeli nqaku zingafumaneka kwi-intanethi: https://www.frontiersin.org/articles/10.3389/frmbi.2024.1451735/full#supplementary-material
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Ixesha leposi: Epreli-18-2025