Ua hōʻike ʻia ka waikawa Propionic (PPA), kahi mea antifungal a me ka mea hoʻohui meaʻai maʻamau, e hoʻoulu ai i ka neurodevelopment maʻamau i nā ʻiole i hele pū ʻia me ka hana ʻino o ka gastrointestinal, kahi i hiki ke kumu ʻia e ka dysbiosis gut. Ua manaʻo ʻia kahi pilina ma waena o ka hōʻike ʻana o ka PPA meaʻai a me ka dysbiosis microbiota gut, akā ʻaʻole i noiʻi pololei ʻia. Maanei, ua noiʻi mākou i nā loli e pili ana i ka PPA i ka haku mele ʻana o ka microbiota gut e alakaʻi ai i ka dysbiosis. Ua hoʻonohonoho ʻia nā microbiomes gut o nā ʻiole i hānai ʻia i kahi meaʻai i mālama ʻole ʻia (n = 9) a me kahi meaʻai i hoʻonui ʻia i ka PPA (n = 13) me ka hoʻohana ʻana i ka hoʻonohonoho metagenomic lōʻihi e loiloi i nā ʻokoʻa i ka haku mele microbial a me nā ala metabolic bacteria. Ua pili ka PPA meaʻai me ka hoʻonui ʻana i ka nui o nā taxa koʻikoʻi, me kekahi mau ʻano Bacteroides, Prevotella, a me Ruminococcus, nā lālā i pili mua i ka hana PPA. Loaʻa i nā microbiomes o nā ʻiole i hōʻike ʻia e PPA nā ala hou aʻe e pili ana i ka metabolism lipid a me ka biosynthesis hormone steroid. Hōʻike kā mākou mau hopena hiki i ka PPA ke hoʻololi i ka microbiota gut a me kona mau ala metabolic pili. Hōʻike kēia mau loli i ʻike ʻia e hiki i nā mea hoʻomalu i hoʻokaʻawale ʻia he palekana no ka ʻai ke hoʻololi i ka haku mele ʻana o ka microbiota ʻōpū, a ma ka huli ʻana, i ke olakino kanaka.
Ua kapa pinepine ʻia ka microbiome kanaka ʻo "ke kino hope loa o ke kino" a he kuleana koʻikoʻi kona i ke olakino kanaka (Baquero lāua ʻo Nombela, 2012). ʻO ka mea kūikawā, ua ʻike ʻia ka microbiome ʻōpū no kona mana ākea a me kāna kuleana i nā hana koʻikoʻi he nui. Nui nā bacteria commensal i loko o ka ʻōpū, e noho ana i nā niches kaiaola he nui, e hoʻohana ana i nā meaʻai, a e hoʻokūkū ana me nā pathogens hiki (Jandhyala et al., 2015). Hiki i nā ʻāpana bacteria like ʻole o ka microbiota ʻōpū ke hana i nā meaʻai koʻikoʻi e like me nā huaora a me ka hoʻolaha ʻana i ka ʻai ʻana (Rowland et al., 2018). Ua hōʻike ʻia hoʻi nā metabolites bacteria e hoʻohuli i ka ulu ʻana o nā ʻiʻo a hoʻomaikaʻi i nā ala metabolic a me nā ala pale (Heijtz et al., 2011; Yu et al., 2022). He ʻokoʻa loa ka haku mele ʻana o ka microbiome ʻōpū kanaka a hilinaʻi ia i nā kumu genetic a me ke kaiapuni e like me ka ʻai, ke kāne, nā lāʻau lapaʻau, a me ke kūlana olakino (Kumbhare et al., 2019).
ʻO ka ʻai a ka makuahine kahi mea koʻikoʻi o ka ulu ʻana o ka fetal a me ka neonatal a he kumu paha o nā hui e hiki ke hoʻohuli i ka ulu ʻana (Bazer et al., 2004; Innis, 2014). ʻO kekahi hui hoihoi, ʻo ia ka propionic acid (PPA), kahi huahana momona pōkole i loaʻa mai ka fermentation bacteria a me kahi mea hoʻohui meaʻai (den Besten et al., 2013). Loaʻa i ka PPA nā waiwai antibacterial a me antifungal a no laila hoʻohana ʻia ma ke ʻano he mea mālama meaʻai a ma nā noi ʻoihana e kāohi i ka ulu ʻana o ka mold a me ka bacteria (Wemmenhove et al., 2016). He ʻokoʻa nā hopena o ka PPA ma nā ʻiʻo like ʻole. I loko o ke akepaʻa, loaʻa i ka PPA nā hopena anti-inflammatory ma ka hoʻopilikia ʻana i ka hōʻike ʻana o ka cytokine i nā macrophages (Kawasoe et al., 2022). Ua ʻike ʻia kēia hopena hoʻoponopono i nā cell immune ʻē aʻe, e alakaʻi ana i ka downregulation o ka mumū (Haase et al., 2021). Eia naʻe, ua ʻike ʻia ka hopena ʻē aʻe i loko o ka lolo. Ua hōʻike nā haʻawina mua e hoʻoulu ka hōʻike ʻana o PPA i ka hana like me ka autism i nā ʻiole (El-Ansary et al., 2012). Ua hōʻike ʻia nā haʻawina ʻē aʻe e hiki i ka PPA ke hoʻoulu i ka gliosis a hoʻāla i nā ala pro-inflammatory i loko o ka lolo (Abdelli et al., 2019). No ka mea he waikawa nāwaliwali ka PPA, hiki iā ia ke hoʻolaha ma o ka epithelium ʻōpū i loko o ke kahe koko a pēlā e hele ai i nā pale palena e pili ana i ka pale koko-lolo a me ka placenta (Stinson et al., 2019), e hōʻike ana i ke koʻikoʻi o ka PPA ma ke ʻano he metabolite hoʻoponopono i hana ʻia e ka bacteria. ʻOiai ke noiʻi ʻia nei ke kuleana hiki o ka PPA ma ke ʻano he kumu pilikia no ka autism, ʻo kona hopena i nā poʻe me ka autism e ʻoi aku ma mua o ka hoʻoulu ʻana i ka hoʻokaʻawale neural.
ʻO nā hōʻailona Gastrointestinal e like me ka maʻi diarrhea a me ka constipation he mea maʻamau i nā poʻe maʻi me nā maʻi neurodevelopmental (Cao et al., 2021). Ua hōʻike ʻia nā haʻawina mua he ʻokoʻa ka microbiome o nā poʻe maʻi me nā maʻi autism spectrum (ASD) mai ko nā kānaka olakino, e hōʻike ana i ke alo o ka gut microbiota dysbiosis (Finegold et al., 2010). Pēlā nō, ʻokoʻa hoʻi nā ʻano microbiome o nā poʻe maʻi me nā maʻi mumū o ka ʻōpū, ka momona, ka maʻi Alzheimer, a me nā mea ʻē aʻe mai ko nā kānaka olakino (Turnbaugh et al., 2009; Vogt et al., 2017; Henke et al., 2019). Eia naʻe, a hiki i kēia lā, ʻaʻohe pilina kumu i hoʻokumu ʻia ma waena o ka gut microbiome a me nā maʻi neurological a i ʻole nā ​​​​hōʻailona (Yap et al., 2021), ʻoiai ua manaʻo ʻia he nui nā ʻano bacteria e pāʻani i kahi kuleana i kekahi o kēia mau kūlana maʻi. No ka laʻana, ʻoi aku ka nui o Akkermansia, Bacteroides, Clostridium, Lactobacillus, Desulfovibrio a me nā genera ʻē aʻe i loko o ka microbiota o nā maʻi me ka autism (Tomova et al., 2015; Golubeva et al., 2017; Cristiano et al., 2018; Zurita et al., 2020). ʻIke ʻia, ʻike ʻia nā ʻano lālā o kekahi o kēia mau genera he mau genes e pili ana i ka hana ʻana o PPA (Reichardt et al., 2014; Yun lāua ʻo Lee, 2016; Zhang et al., 2019; Baur lāua ʻo Dürre, 2023). Hāʻawi ʻia i nā waiwai antimicrobial o PPA, ʻo ka hoʻonui ʻana i kona nui he mea maikaʻi paha no ka ulu ʻana o nā bacteria hana PPA (Jacobson et al., 2018). No laila, hiki i kahi kaiapuni waiwai PFA ke alakaʻi i nā loli i ka microbiota ʻōpū, me nā pathogens gastrointestinal, hiki ke lilo i mau kumu kūpono e alakaʻi ai i nā hōʻailona gastrointestinal.
ʻO kahi nīnau nui i ka noiʻi microbiome, inā paha he kumu a hōʻailona paha nā ʻokoʻa o ke ʻano microbial o nā maʻi kumu. ʻO ka hana mua i ka wehewehe ʻana i ka pilina paʻakikī ma waena o ka ʻai, ka microbiome gut, a me nā maʻi neurological, ʻo ia ka loiloi ʻana i nā hopena o ka ʻai ma ke ʻano microbial. No kēia hopena, ua hoʻohana mākou i ka heluhelu lōʻihi ʻana o ka metagenomic sequencing e hoʻohālikelike i nā microbiomes gut o nā keiki a nā ʻiole i hānai ʻia i ka ʻai momona PPA a i ʻole PPA-depleted. Ua hānai ʻia nā keiki i ka ʻai like me ko lākou mau makuahine. Ua kuhi mākou he hopena ka ʻai momona PPA i nā loli i ke ʻano microbial gut a me nā ala hana microbial, ʻoiai nā mea e pili ana i ka metabolism PPA a/a i ʻole ka hana PPA.
Ua hoʻohana kēia haʻawina i nā ʻiole transgenic FVB/N-Tg(GFAP-GFP)14Mes/J (Jackson Laboratories) i hoʻonui i ka protein fluorescent ʻōmaʻomaʻo (GFP) ma lalo o ka mana o ka mea hoʻolaha GFAP glia-specific e hahai ana i nā alakaʻi o ke Komite Mālama a me ka Hoʻohana ʻana i nā Holoholona o ke Kulanui o Central Florida (UCF-IACUC) (Helu ʻAe Hoʻohana Holoholona: PROTO202000002). Ma hope o ka hoʻopau ʻana i ka waiū, ua hoʻokomo ʻia nā ʻiole i loko o nā hale paʻa me 1-5 mau ʻiole o kēlā me kēia wahine i kēlā me kēia hale paʻa. Ua hānai ʻia nā ʻiole ad libitum me kahi papaʻai hoʻomalu i hoʻomaʻemaʻe ʻia (ʻai maʻamau open-label i hoʻololi ʻia, 16 kcal% momona) a i ʻole kahi papaʻai i hoʻohui ʻia me ka sodium propionate (ʻai maʻamau open-label i hoʻololi ʻia, 16 kcal% momona, nona ka 5,000 ppm sodium propionate). ʻO ka nui o ka sodium propionate i hoʻohana ʻia ua like ia me 5,000 mg PFA/kg kaumaha o ka meaʻai. ʻO kēia ka nui kiʻekiʻe loa o ka PPA i ʻae ʻia no ka hoʻohana ʻana ma ke ʻano he mea mālama meaʻai. No ka hoʻomākaukau ʻana no kēia haʻawina, ua hānai ʻia nā ʻiole makua i nā meaʻai ʻelua no 4 mau pule ma mua o ka male ʻana a ua hoʻomau ʻia i ka wā hāpai o ka makuahine. Ua hoʻōki ʻia nā ʻiole mamo [22 ʻiole, 9 mau kaohi (6 kāne, 3 wahine) a me 13 PPA (4 kāne, 9 wahine)] a laila hoʻomau ʻia i ka ʻai like me nā makuahine no 5 mau mahina. Ua pepehi ʻia nā ʻiole mamo i 5 mau mahina o ka makahiki a ua hōʻiliʻili ʻia ko lākou mau ʻōpū a mālama mua ʻia i loko o nā ʻōmole microcentrifuge 1.5 ml ma -20°C a laila ua hoʻoili ʻia i loko o kahi pahu hau -80°C a hiki i ka pau ʻana o ka DNA kikowaena a ua unuhi ʻia nā waikawa nucleic microbial.
Ua wehe ʻia ka DNA kikowaena e like me kahi protocol i hoʻololi ʻia (Charalampous et al., 2019). I ka pōkole, ua hoʻoili ʻia nā mea o loko o ka fecal i 500 µl InhibitEX (Qiagen, Cat#/ID: 19593) a mālama ʻia i loko o ka hau. Hana i ka nui o 1-2 mau pellets fecal no kēlā me kēia unuhi. A laila ua hoʻohuihui ʻia nā mea o loko o ka fecal me ka hoʻohana ʻana i kahi pestle plastik i loko o ka paipu e hana i kahi slurry. E centrifuge i nā laʻana ma 10,000 RCF no 5 min a hiki i ka pelleting ʻana o nā laʻana, a laila e omo i ka supernatant a hoʻokuʻu hou i ka pellet i loko o 250 µl 1 × PBS. E hoʻohui i 250 µl 4.4% saponin solution (TCI, helu huahana S0019) i ka laʻana ma ke ʻano he detergent e wehe ai i nā membrane cell eukaryotic. Ua hui mālie ʻia nā laʻana a hiki i ka laumania a hoʻomoʻa ʻia ma ka mahana o ka lumi no 10 min. ʻO ka mea aʻe, no ka hoʻopilikia ʻana i nā pūnaewele eukaryotic, ua hoʻohui ʻia he 350 μl wai nuclease-free i ka laʻana, hoʻomoʻa ʻia no 30 kekona, a laila ua hoʻohui ʻia he 12 μl 5 M NaCl. A laila ua centrifuged ʻia nā laʻana ma 6000 RCF no 5 min. E omo i ka supernatant a hoʻokuʻu hou i ka pellet i loko o 100 μl 1X PBS. No ka wehe ʻana i ka DNA kikowaena, e hoʻohui i ka 100 μl HL-SAN buffer (12.8568 g NaCl, 4 ml 1M MgCl2, 36 ml wai nuclease-free) a me 10 μl HL-SAN enzyme (ArticZymes P/N 70910-202). Ua hui maikaʻi ʻia nā laʻana ma o ka pipetting a hoʻomoʻa ʻia ma 37 °C no 30 min ma 800 rpm ma kahi Eppendorf™ ThermoMixer C. Ma hope o ka hoʻomoʻa ʻana, ua centrifuged ʻia ma 6000 RCF no 3 min a holoi ʻia i ʻelua manawa me 800 µl a me 1000 µl 1X PBS. ʻO ka hope loa, e hoʻokuʻu hou i ka pellet i loko o 100 µl 1X PBS.
Ua hoʻokaʻawale ʻia ka DNA bacteria holoʻokoʻa me ka hoʻohana ʻana i ka New England Biolabs Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, Cat# T3010L). Ua hoʻololi iki ʻia ke kaʻina hana maʻamau i hāʻawi ʻia me ka pahu. E hoʻomoʻa a mālama i ka wai nuclease-free ma 60°C ma mua o ka hana no ka elution hope loa. E hoʻohui i 10 µl Proteinase K a me 3 µl RNase A i kēlā me kēia laʻana. A laila e hoʻohui i 100 µl Cell Lysis Buffer a hui mālie. A laila ua hoʻomoʻa ʻia nā laʻana i loko o kahi Eppendorf™ ThermoMixer C ma 56°C a me 1400 rpm no ka liʻiliʻi he 1 hola a hiki i 3 hola. Ua centrifuged ʻia nā laʻana i hoʻomoʻa ʻia ma 12,000 RCF no 3 mau minuke a ua hoʻoili ʻia ka supernatant mai kēlā me kēia laʻana i kahi ʻōmole microcentrifuge 1.5 mL ʻokoʻa e loaʻa ana he 400 µL o ka hopena nakinaki. A laila ua pulse vortexed ʻia nā ʻōmole no 5-10 kekona ma 1 kekona mau manawa. E hoʻoili i ka wai holoʻokoʻa o kēlā me kēia hāpana (ma kahi o 600–700 µL) i kahi kāwele kānana i kau ʻia i loko o kahi paipu hōʻiliʻili kahe. Ua centrifuged ʻia nā paipu ma 1,000 RCF no 3 mau minuke e ʻae i ka hoʻopaʻa mua ʻana o ka DNA a laila centrifuged ʻia ma 12,000 RCF no 1 minuke e wehe i ke koena wai. Ua hoʻoili ʻia ke kolamu hāpana i kahi paipu hōʻiliʻili hou a laila holoi ʻia i ʻelua manawa. No ka holoi mua ʻana, e hoʻohui i 500 µL o ka buffer holoi i kēlā me kēia paipu. E hoʻohuli i ka paipu 3-5 mau manawa a laila centrifuge ma 12,000 RCF no 1 minuke. E hoʻolei i ka wai mai ka paipu hōʻiliʻili a hoʻihoʻi i ka kāwele kānana i loko o ka paipu hōʻiliʻili like. No ka holoi ʻelua, e hoʻohui i 500 µL o ka buffer holoi i ke kānana me ka ʻole o ka hoʻohuli ʻana. Ua centrifuged ʻia nā hāpana ma 12,000 RCF no 1 minuke. E hoʻoili i ke kānana i kahi ʻōmole LoBind® 1.5 mL a hoʻohui i 100 µL o ka wai nuclease-free i hoʻomehana mua ʻia. Ua hoʻomoʻa ʻia nā kānana ma ka mahana o ka lumi no 1 minuke a laila centrifuged ma 12,000 RCF no 1 minuke. Ua mālama ʻia ka DNA i hoʻoheheʻe ʻia ma -80°C.
Ua helu ʻia ka nui o ka DNA me ka hoʻohana ʻana i kahi Qubit™ 4.0 Fluorometer. Ua hoʻomākaukau ʻia ka DNA me ka hoʻohana ʻana i ka Qubit™ 1X dsDNA High Sensitivity Kit (Cat. No. Q33231) e like me nā kuhikuhi a ka mea hana. Ua ana ʻia ka hoʻolaha ʻana o ka lōʻihi o ka ʻāpana DNA me ka hoʻohana ʻana i kahi Aglient™ 4150 a i ʻole 4200 TapeStation. Ua hoʻomākaukau ʻia ka DNA me ka hoʻohana ʻana i ka Agilent™ Genomic DNA Reagents (Cat. No. 5067-5366) a me Genomic DNA ScreenTape (Cat. No. 5067-5365). Ua hana ʻia ka hoʻomākaukau ʻana o ka waihona puke me ka hoʻohana ʻana i ka Oxford Nanopore Technologies™ (ONT) Rapid PCR Barcoding Kit (SQK-RPB004) e like me nā kuhikuhi a ka mea hana. Ua hoʻonohonoho ʻia ka DNA me ka hoʻohana ʻana i kahi ONT GridION™ Mk1 sequencer me kahi Min106D flow cell (R 9.4.1). ʻO nā hoʻonohonoho hoʻonohonoho: ke kāhea kumu pololei kiʻekiʻe, ka waiwai q liʻiliʻi loa o 9, ka hoʻonohonoho barcode, a me ka ʻoki barcode. Ua hoʻopaʻa ʻia nā laʻana no 72 mau hola, a ma hope iho ua waiho ʻia ka ʻikepili kelepona kumu no ka hana hou aku a me ka nānā ʻana.
Ua hana ʻia ka hana ʻana o Bioinformatics me ka hoʻohana ʻana i nā ʻano hana i wehewehe mua ʻia (Greenman et al., 2024). Ua māhele ʻia nā faila FASTQ i loaʻa mai ka sequencing i nā papa kuhikuhi no kēlā me kēia laʻana. Ma mua o ka nānā ʻana o ka bioinformatics, ua hana ʻia ka ʻikepili me ka hoʻohana ʻana i ka pipeline aʻe: ʻo ka mua, ua hoʻohui ʻia nā faila FASTQ o nā laʻana i loko o hoʻokahi faila FASTQ. A laila, ua kānana ʻia nā heluhelu ma lalo o 1000 bp me ka hoʻohana ʻana iā Filtlong v. 0.2.1, me ka hoʻololi wale ʻana o ka parameter –min_length 1000 (Wick, 2024). Ma mua o ka kānana hou ʻana, ua kāohi ʻia ka maikaʻi o ka heluhelu me ka hoʻohana ʻana iā NanoPlot v. 1.41.3 me nā parameter aʻe: –fastq –plots dot –N50 -o(De Coster lāua ʻo Rademakers, 2023). Ua hoʻohālikelike ʻia nā heluhelu i ka genome kuhikuhi ʻiole GRCm39 (GCF_000001635.27) me ka hoʻohana ʻana iā minimap2 v. 2.24-r1122 e wehe i nā heluhelu i haumia ʻia e ka mea hoʻokipa me nā palena aʻe: -L -ax map-ont(Lee, 2018). Ua hoʻololi ʻia nā faila hoʻonohonoho i hana ʻia i ke ʻano BAM me ka hoʻohana ʻana i ka samtools view -b (Danecek et al., 2021) ma samtools v. 1.16.1. A laila ua ʻike ʻia nā heluhelu i hoʻonohonoho ʻole ʻia me ka hoʻohana ʻana i ka samtools view -b -f 4, e hōʻike ana ʻaʻole no ka genome kikowaena kēia mau heluhelu. Ua hoʻololi ʻia nā heluhelu i hoʻonohonoho ʻole ʻia i ke ʻano FASTQ me ka hoʻohana ʻana i ka samtools bam2fq me nā palena paʻamau. Ua holo hou ʻia ʻo NanoPlot ma nā heluhelu i kānana hou ʻia me ka hoʻohana ʻana i nā hoʻonohonoho i wehewehe ʻia ma mua. Ma hope o ka kānana ʻana, ua hōʻuluʻulu ʻia nā ʻikepili metagenomic me ka hoʻohana ʻana i ka metaflye v. 2.8.2-b1689 me nā palena penei: –nano-raw–meta (Kolmogorov et al., 2020). E waiho i nā palena i koe ma ko lākou mau waiwai paʻamau. Ma hope o ka hōʻuluʻulu ʻana, ua hoʻopaʻa ʻia nā heluhelu i kānana ʻia i ka hōʻuluʻulu me ka hoʻohana ʻana i ka minimap2, a ua hoʻohana ʻia ke palena -ax map-ont e hana i kahi faila hoʻonohonoho ma ke ʻano SAM. Ua hoʻomaʻemaʻe mua ʻia ka hōʻuluʻulu ʻana me ka hoʻohana ʻana i ka racon v. 1.4.20 me nā palena penei: -m 8 -x -6 -g -8 -w 500 -u (Vaser et al., 2017). Ma hope o ka pau ʻana o ka racon, ua hoʻomaʻemaʻe hou ʻia me medaka v. 1.7.2, me ka hoʻohana ʻana i ka medaka_consesus, me nā palena āpau koe wale nō ka palena -m i waiho ʻia ma ko lākou mau waiwai paʻamau. Ua hoʻonohonoho ʻia ke palena -m i r941_min_hac_g507 e kuhikuhi i ke kemika o ke kahe ʻana o ke kelepona a me ke kāhea kumu pololei kiʻekiʻe i hoʻohana ʻia no kā mākou ʻikepili (nanoporetech/medaka, 2024). Ua hoʻohana ʻia ka ʻikepili i kānana ʻia (ma hope aku i kapa ʻia ʻo ka ʻikepili microbial) a me ka hōʻuluʻulu hope loa i hoʻomaʻemaʻe ʻia no ka nānā ʻana ma hope.
No ka hoʻokaʻawale taxonomic, ua hoʻokaʻawale ʻia nā heluhelu a me nā contigs i hōʻuluʻulu ʻia me ka hoʻohana ʻana iā Kraken2 v. 2.1.2 (Wood et al., 2019). Hoʻopuka i nā hōʻike a hoʻopuka i nā faila no nā heluhelu a me nā ʻākoakoa, kēlā me kēia. E hoʻohana i ke koho –use-names e kālailai i nā heluhelu a me nā ʻākoakoa. Ua kuhikuhi ʻia nā koho –gzip-compressed a me –paired no nā ʻāpana heluhelu. Ua kuhi ʻia ka nui o nā taxa i loko o nā metagenomes me ka hoʻohana ʻana iā Bracken v. 2.8 (Lu et al., 2017). Ua hana mua mākou i kahi waihona ʻikepili kmer e loaʻa ana he 1000 mau kumu me ka hoʻohana ʻana i ka bracken-build me nā palena penei: -d-k 35 -l 1000 Ke kūkulu ʻia, holo ka bracken ma muli o ka hōʻike i hana ʻia e kraken2 a kānana i ka ʻikepili me ka hoʻohana ʻana i nā koho aʻe: -d -I -O-p 1000 -l

Ma waena o lākou, ua koho ʻia ʻo P, G a i ʻole S ma muli o ka pae hoʻokaʻawale e kālailai ʻia nei. No ka hoʻēmi ʻana i ka hopena o nā hoʻokaʻawale maikaʻi ʻole, ua hoʻohana ʻia kahi paepae nui o 1e-4 (1/10,000 heluhelu). Ma mua o ka loiloi helu, ua hoʻololi ʻia nā nui o ka nui i hōʻike ʻia e Bracken (fraction_total_reads) me ka hoʻohana ʻana i ka hoʻololi ʻana o ka log-ratio centered (CLR) (Aitchison, 1982). Ua koho ʻia ke ʻano CLR no ka hoʻololi ʻikepili no ka mea he invariant unahi a lawa ia no nā ʻikepili ʻaʻole sparse (Gloor et al., 2017). Hoʻohana ka hoʻololi ʻana o CLR i ka logarithm kūlohelohe. Ua hoʻonohonoho ʻia ka ʻikepili helu i hōʻike ʻia e Bracken me ka hoʻohana ʻana i ka hōʻike log pili (RLE) (Anders lāua ʻo Huber, 2010). Ua hana ʻia nā helu me ka hoʻohana ʻana i ka hui pū ʻana o matplotlib v. 3.7.1, seaborn v. 3.7.2 a me nā logarithms sequential (Gloor et al., 2017). 0.12.2 a me nā instantanotations v. 0.5.0 (Hunter, 2007; Waskom, 2021; Charlier et al., 2022). Ua helu ʻia ka lakio Bacillus/Bacteroidetes no kēlā me kēia laʻana me ka hoʻohana ʻana i nā helu bacteria i hoʻonohonoho ʻia. Ua hoʻopuni ʻia nā waiwai i hōʻike ʻia ma nā papa i 4 mau wahi decimal. Ua helu ʻia ka helu kuhikuhi ʻokoʻa Simpson me ka hoʻohana ʻana i ka script alpha_diversity.py i hāʻawi ʻia ma ka pūʻolo KrakenTools v. 1.2 (Lu et al., 2022). Hāʻawi ʻia ka hōʻike Bracken ma ka script a ua hāʻawi ʻia ka helu kuhikuhi Simpson "Si" no ka parameter -an. Ua wehewehe ʻia nā ʻokoʻa koʻikoʻi i ka nui ma ke ʻano he ʻokoʻa CLR awelika ≥ 1 a i ʻole ≤ -1. ʻO ka ʻokoʻa CLR awelika o ±1 e hōʻike ana i ka piʻi ʻana o 2.7-fold i ka nui o kahi ʻano laʻana. Hōʻike ka hōʻailona (+/-) inā ʻoi aku ka nui o ka taxon ma ka laʻana PPA a me ka laʻana kaohi, kēlā me kēia. Ua hoʻoholo ʻia ke koʻikoʻi me ka hoʻohana ʻana i ka hoʻāʻo Mann-Whitney U (Virtanen et al., 2020). Ua hoʻohana ʻia ʻo Statsmodels v. 0.14 (Benjamini lāua ʻo Hochberg, 1995; Seabold lāua ʻo Perktold, 2010), a ua hoʻopili ʻia ke kaʻina hana Benjamini-Hochberg e hoʻoponopono no nā hoʻāʻo he nui. Ua hoʻohana ʻia kahi waiwai p i hoʻoponopono ʻia ≤ 0.05 ma ke ʻano he paepae no ka hoʻoholo ʻana i ke koʻikoʻi helu.
Ua hana ʻia ka hoʻākāka ʻana o nā gene a me ka helu ʻana o ka nui o ka nui me ka hoʻohana ʻana i kahi mana i hoʻololi ʻia o ke kaʻina hana i wehewehe ʻia e Maranga et al. (Maranga et al., 2023). ʻO ka mea mua, ua wehe ʻia nā contigs pōkole ma mua o 500 bp mai nā ʻākoakoa āpau me ka hoʻohana ʻana iā SeqKit v. 2.5.1 (Shen et al., 2016). A laila ua hoʻohui ʻia nā ʻākoakoa i koho ʻia i loko o kahi pan-metagenome. Ua ʻike ʻia nā kiʻi heluhelu hāmama (ORFs) me ka hoʻohana ʻana iā Prodigal v. 1.0.1 (kahi mana like o Prodigal v. 2.6.3) me nā palena penei: -d-f gff-i -O-T 24 -p meta -C 10000 (Hyett et al., 2012; Jaenicke, 2024). A laila ua kānana ʻia nā faila nucleotide i loaʻa me ka hoʻohana ʻana iā Python e wehe i nā genes piha ʻole. A laila ua hoʻohana ʻia ʻo CD-HIT v. 4.8.1 e hoʻohui i nā genes me nā palena penei: cd-hit-est -i -O-c 0.95 -s 0.85 -aS 0.9 -n 10 -d 256 -M 350000 -T 24 -l 100 -g 1 (Fu et al., 2012). Ua hoʻohana ʻia ka papa inoa gene non-redundant i hana ʻia e kuhi i ka nui o ka gene a me ka annotation. Ua kuhi ʻia ka nui o ka gene pili me ka hoʻohana ʻana iā KMA v. 1.4.9 (Clausen et al., 2018). ʻO ka mea mua, e hana i kahi faila index me ka hoʻohana ʻana i ka index KMA me nā palena penei: -i -OA laila, me ka hoʻohana ʻana i ka papa kuhikuhi i hana ʻia me nā heluhelu microbial no kēlā me kēia laʻana e like me ka mea i wehewehe ʻia ma ka ʻāpana Bioinformatics Pipeline, ua holo ʻia ʻo KMA me nā palena penei: -i -O-t_db-bcNano -bc 0.7 -ef -t 24. A laila, ua hoʻohālikelike ʻia nā helu gene me ka hoʻohana ʻana iā CLR, a ua hoʻohana ʻia ka papa loiloi ʻāpana nui (PCA) o Sci-kit learn (Pedregosa et al., 2011). Ua hana ʻia ka annotation gene i wānana ʻia ma ka papa inoa gene non-redundant me ka hoʻohana ʻana i ka script emapper.py o eggNOG v. 2.1.12 a me ka mana waihona ʻikepili eggNOG 5.0.2 me nā palena penei: –typepe CDS –cpu 24 -i– Papa inoa ʻikepili–go_evidence ʻAʻole uila – hoʻopuka– Papa kuhikuhi hoʻopuka–target_orthologs āpau –seed_ortholog_evalue 0.001 –seed_ortholog_score 60 –query_cover 20 –subject_cover 0 –translate –override –temp_dir(Cantalapiedra et al., 2021). Ua nānā ʻia nā hopena KMA e koho i nā genes me ka uhi template lawa a me ka ʻike template (≥ 90%) a me ka nui (hohonu ≥ 3). Ua hoʻololi ʻia nā hopena hohonu KMA me ka hoʻohana ʻana iā CLR e like me ka mea i wehewehe ʻia ma luna. A laila ua hoʻohālikelike ʻia nā hopena KMA me nā ID contig mai nā hopena annotation hana a me ka hoʻokaʻawale ʻana me ka hoʻohana ʻana i ke kumu contig no kēlā me kēia gene. E like me nā taxa, ua wehewehe ʻia nā ʻokoʻa koʻikoʻi i ka nui o ka gene ma ke ʻano he mau genes me ka ʻokoʻa CLR awelika ≥ 1 a i ʻole ≤ -1, me kahi hōʻailona (+/-) e hōʻike ana ua nui ka gene ma nā PPA a i ʻole nā ​​​​laʻana kaohi, kēlā me kēia.
Ua hoʻokaʻawale mua ʻia nā genes e like me ka Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO) identifiers i hāʻawi ʻia e eggNOG e hoʻohālikelike i nā nui o ke ala gene. Ua wehe ʻia nā genes me ka ʻole o nā knockouts a i ʻole nā ​​​​genes me nā knockouts he nui ma mua o ka nānā ʻana. A laila ua helu ʻia ka nui o kēlā me kēia KO i kēlā me kēia laʻana a ua hana ʻia ka nānā ʻana i ka helu. Ua wehewehe ʻia nā genes metabolism PPA e like me kekahi gene i hāʻawi ʻia i kahi lālani ko00640 ma ke kolamu KEGG_Pathway, e hōʻike ana i kahi kuleana i ka metabolism propionate e like me KEGG. Ua helu ʻia nā genes i ʻike ʻia e pili ana i ka hana PPA ma ka Papa Hoʻohui 1 (Reichardt et al., 2014; Yang et al., 2017). Ua hana ʻia nā hoʻāʻo Permutation e ʻike i ka metabolism PPA a me nā genes hana i ʻoi aku ka nui ma kēlā me kēia ʻano laʻana. Ua hana ʻia hoʻokahi tausani permutations no kēlā me kēia gene i kālailai ʻia. Ua hoʻohana ʻia kahi p-value o 0.05 ma ke ʻano he cutoff e hoʻoholo ai i ke koʻikoʻi helu. Ua hāʻawi ʻia nā annotations hana i nā genes pākahi i loko o kahi hui e pili ana i nā annotations o nā genes ʻelele i loko o ka hui. Hiki ke ʻike ʻia nā taxa e pili ana me ka metabolism PPA a/a i ʻole ka hana PPA ma ka hoʻohālikelike ʻana i nā ID contig i nā faila hoʻopuka Kraken2 me nā ID contig like i mālama ʻia i ka wā o ka annotation hana me ka hoʻohana ʻana i ka eggNOG. Ua hana ʻia ka hoʻāʻo koʻikoʻi me ka hoʻohana ʻana i ka hoʻāʻo Mann-Whitney U i wehewehe mua ʻia. Ua hana ʻia ka hoʻoponopono no nā hoʻāʻo he nui me ka hoʻohana ʻana i ke kaʻina hana Benjamini-Hochberg. Ua hoʻohana ʻia kahi p-value o ≤ 0.05 ma ke ʻano he cutoff e hoʻoholo ai i ke koʻikoʻi helu.
Ua loiloi ʻia ke ʻano like ʻole o ka microbiome ʻōpū o nā ʻiole me ka hoʻohana ʻana i ka helu kuhikuhi ʻokoʻa Simpson. ʻAʻohe ʻokoʻa koʻikoʻi i ʻike ʻia ma waena o nā laʻana kaohi a me nā PPA ma ke ʻano o ka ʻokoʻa o ka genus a me nā ʻano (p-value no ka genus: 0.18, p-value no nā ʻano: 0.16) (Kiʻi 1). A laila ua hoʻohālikelike ʻia ka haku mele microbial me ka hoʻohana ʻana i ka loiloi ʻāpana nui (PCA). Hōʻike ka Kiʻi 2 i ka hui ʻana o nā laʻana e kā lākou phyla, e hōʻike ana he ʻokoʻa nā ʻano o nā microbiomes ma waena o ka PPA a me nā laʻana kaohi. ʻAʻole i ʻike nui ʻia kēia hui ʻana ma ka pae genus, e hōʻike ana e hoʻopilikia ana ka PPA i kekahi mau bacteria (Kiʻi Hoʻohui 1).
Kiʻi 1. ʻO ke ʻano like ʻole o ka genera a me nā ʻano o ka microbiome ʻōpū o ka ʻiole. Nā kiʻi pahu e hōʻike ana i nā ʻōkuhi ʻokoʻa Simpson o ka genera (A) a me nā ʻano (B) ma PPA a me nā laʻana kaohi. Ua hoʻoholo ʻia ke koʻikoʻi me ka hoʻohana ʻana i ka hoʻāʻo Mann-Whitney U, a ua hana ʻia ka hoʻoponopono pinepine me ka hoʻohana ʻana i ke kaʻina hana Benjamini-Hochberg. ns, ʻaʻole koʻikoʻi ka waiwai-p (p>0.05).
Kiʻi 2. Nā hopena o ka loiloi ʻāpana nui o ka haku mele microbiome o ka ʻiole ma ka pae ʻano. Hōʻike ka pakuhi loiloi ʻāpana nui i ka hoʻolaha ʻana o nā laʻana ma o kā lākou mau ʻāpana nui ʻelua mua. Hōʻike nā kala i ke ʻano laʻana: he poni nā ʻiole i hōʻike ʻia e PPA a he melemele nā ​​ʻiole kaohi. Ua hoʻolālā ʻia nā ʻāpana nui 1 a me 2 ma ka axis-x a me ka axis-y, kēlā me kēia, a ua hōʻike ʻia ma ke ʻano he lakio ʻokoʻa i wehewehe ʻia.
Ma ka hoʻohana ʻana i ka ʻikepili helu i hoʻololi ʻia e RLE, ua ʻike ʻia ka emi nui ʻana o ka lakio waena o Bacteroidetes/Bacilli i nā ʻiole kaohi a me PPA (kaohi: 9.66, PPA: 3.02; p-value = 0.0011). ʻO kēia ʻokoʻa ma muli o ka nui o Bacteroidetes i nā ʻiole PPA i hoʻohālikelike ʻia me nā kaohi, ʻoiai ʻaʻole koʻikoʻi ka ʻokoʻa (kaohi awelika CLR: 5.51, PPA awelika CLR: 6.62; p-value = 0.054), ʻoiai ua like ka nui o Bacteroidetes (kaohi awelika CLR: 7.76, PPA awelika CLR: 7.60; p-value = 0.18).
Ua hōʻike ka loiloi o ka nui o nā lālā taxonomic o ka microbiome gut he 1 phylum a me 77 mau ʻano i ʻokoʻa nui ma waena o nā laʻana PPA a me nā kaohi (Papa Hoʻohui 2). ʻOi aku ka nui o 59 mau ʻano i loko o nā laʻana PPA ma mua o nā laʻana kaohi, ʻoiai ʻoi aku ka nui o 16 mau ʻano wale nō i loko o nā laʻana kaohi ma mua o nā laʻana PPA (Kiʻi 3).
Kiʻi 3. ʻOkoʻa ka nui o nā taxa i loko o ka microbiome ʻōpū o PPA a me nā ʻiole kaohi. Hōʻike nā kiʻi lua pele i nā ʻokoʻa i ka nui o nā genera (A) a i ʻole nā ​​​​ʻano (B) ma waena o PPA a me nā laʻana kaohi. Hōʻike nā kiko hina ʻaʻohe ʻokoʻa koʻikoʻi i ka nui o nā taxa. Hōʻike nā kiko kala i nā ʻokoʻa koʻikoʻi i ka nui (p-value ≤ 0.05). ʻO nā taxa 20 kiʻekiʻe me nā ʻokoʻa nui loa i ka nui ma waena o nā ʻano laʻana e hōʻike ʻia ma ka ʻulaʻula a me ka uliuli māmā (nā laʻana kaohi a me PPA), kēlā me kēia. ʻOi aku ka nui o nā kiko melemele a me ka poni ma ka liʻiliʻi he 2.7 mau manawa i ʻoi aku ka nui ma nā laʻana kaohi a i ʻole PPA ma mua o nā kaohi. Hōʻike nā kiko ʻeleʻele i nā taxa me nā nui like ʻole, me nā ʻokoʻa CLR awelika ma waena o -1 a me 1. Ua helu ʻia nā waiwai P me ka hoʻohana ʻana i ka hoʻāʻo Mann-Whitney U a hoʻoponopono ʻia no ka hoʻāʻo ʻana he nui me ka hoʻohana ʻana i ke kaʻina hana Benjamini-Hochberg. Hōʻike nā ʻokoʻa CLR awelika wiwo ʻole i nā ʻokoʻa koʻikoʻi i ka nui.
Ma hope o ke kālailai ʻana i ka haku mele microbial o ka ʻōpū, ua hana mākou i kahi hōʻike hana o ka microbiome. Ma hope o ke kānana ʻana i nā genes haʻahaʻa, ua ʻike ʻia he 378,355 mau genes kū hoʻokahi ma nā laʻana āpau. Ua hoʻohana ʻia ka nui o kēia mau genes i hoʻololi ʻia no ka loiloi ʻāpana nui (PCA), a ua hōʻike nā hopena i kahi kiʻekiʻe o ka hui ʻana o nā ʻano laʻana e pili ana i kā lākou mau ʻaoʻao hana (Kiʻi 4).
Kiʻi 4. Nā hopena PCA e hoʻohana ana i ka ʻikepili hana o ka microbiome ʻōpū o ka ʻiole. Hōʻike ka pakuhi PCA i ka hoʻolaha ʻana o nā laʻana ma o kā lākou mau ʻāpana nui ʻelua. Hōʻike nā kala i ke ʻano laʻana: he poni nā ʻiole i hōʻike ʻia e PPA a he melemele nā ​​ʻiole kaohi. Ua hoʻolālā ʻia nā ʻāpana nui 1 a me 2 ma ka axis-x a me ka axis-y, kēlā me kēia, a ua hōʻike ʻia ma ke ʻano he lakio ʻokoʻa i wehewehe ʻia.
Ua nānā hou mākou i ka nui o nā knockouts KEGG i nā ʻano laʻana like ʻole. Ua ʻike ʻia he 3648 mau knockouts kū hoʻokahi, ʻo 196 o ia mau mea i ʻoi aku ka nui ma nā laʻana kaohi a ʻo 106 i ʻoi aku ka nui ma nā laʻana PPA (Kiʻi 5). Ua ʻike ʻia he 145 mau genes i nā laʻana kaohi a me 61 mau genes i nā laʻana PPA, me nā ʻano like ʻole. ʻOi aku ka waiwai o nā ala e pili ana i ka metabolism lipid a me aminosugar i nā laʻana PPA (Papa Hoʻohui 3). ʻOi aku ka waiwai o nā ala e pili ana i ka metabolism nitrogen a me nā ʻōnaehana relay sulfur i nā laʻana kaohi (Papa Hoʻohui 3). ʻOi aku ka kiʻekiʻe o ka nui o nā genes e pili ana i ka metabolism aminosugar/nucleotide (ko:K21279) a me ka metabolism inositol phosphate (ko:K07291) i nā laʻana PPA (Kiʻi 5). ʻOi aku ka nui o nā genes o nā laʻana kaohi e pili ana i ka metabolism benzoate (ko:K22270), ka metabolism nitrogen (ko:K00368), a me ka glycolysis/gluconeogenesis (ko:K00131) (Kiʻi 5).
Kiʻi 5. ʻOkoʻa ka nui o nā KO i loko o ka microbiome ʻōpū o ka PPA a me nā ʻiole kaohi. Hōʻike ka palapala lua pele i nā ʻokoʻa i ka nui o nā hui hana (KO). Hōʻike nā kiko hina i nā KO nona ka nui ʻaʻole i ʻokoʻa nui ma waena o nā ʻano laʻana (p-value > 0.05). Hōʻike nā kiko kala i nā ʻokoʻa koʻikoʻi i ka nui (p-value ≤ 0.05). Hōʻike ʻia nā 20 KO me nā ʻokoʻa nui loa i ka nui ma waena o nā ʻano laʻana i ka ʻulaʻula a me ka uliuli māmā, e pili ana i nā laʻana kaohi a me PPA, kēlā me kēia. Hōʻike nā kiko melemele a me ka poni i nā KO i ʻoi aku ka nui ma ka liʻiliʻi he 2.7-fold i nā laʻana kaohi a me PPA, kēlā me kēia. Hōʻike nā kiko ʻeleʻele i nā KO me nā nui ʻokoʻa koʻikoʻi, me nā ʻokoʻa CLR awelika ma waena o -1 a me 1. Ua helu ʻia nā waiwai P me ka hoʻohana ʻana i ka hoʻāʻo Mann-Whitney U a hoʻoponopono ʻia no nā hoʻohālikelike he nui me ka hoʻohana ʻana i ke kaʻina hana Benjamini-Hochberg. Hōʻike ʻo NaN ʻaʻole pili ka KO i kahi ala ma KEGG. Hōʻike nā waiwai ʻokoʻa CLR awelika wiwo ʻole i nā ʻokoʻa koʻikoʻi i ka nui. No ka ʻike kikoʻī e pili ana i nā ala e pili ai nā KO i helu ʻia, e ʻike i ka Papa Hoʻohui 3.
Ma waena o nā genes i hōʻike ʻia, he ʻokoʻa nui ka nui o nā genes 1601 ma waena o nā ʻano laʻana (p ≤ 0.05), me kēlā me kēia gene ma ka liʻiliʻi he 2.7-fold ka nui. No kēia mau genes, ʻoi aku ka nui o 4 mau genes i nā laʻana kaohi a ʻoi aku ka nui o 1597 mau genes i nā laʻana PPA. No ka mea he mau waiwai antimicrobial ko PPA, ua nānā mākou i ka nui o ka metabolism PPA a me nā genes hana ma waena o nā ʻano laʻana. Ma waena o nā genes e pili ana i ka metabolism PPA 1332, ʻoi aku ka nui o 27 mau genes i nā laʻana kaohi a ʻoi aku ka nui o 12 mau genes i nā laʻana PPA. Ma waena o nā genes e pili ana i ka hana PPA 223, ʻoi aku ka nui o 1 gene i nā laʻana PPA. Hōʻike hou aku ka Kiʻi 6A i ka nui o nā genes i komo i ka metabolism PPA, me ka nui o nā laʻana kaohi a me nā nui o ka hopena nui, ʻoiai ʻo ka Kiʻi 6B e hōʻike ana i nā genes pākahi me ka nui o ka nui i ʻike ʻia i nā laʻana PPA.
Kiʻi 6. ʻOkoʻa ka nui o nā genes e pili ana i ka PPA i loko o ka microbiome ʻōpū ʻiole. Hōʻike nā kiʻi lua pele i nā ʻokoʻa o ka nui o nā genes e pili ana me ka metabolism PPA (A) a me ka hana PPA (B). Hōʻike nā kiko hina i nā genes nona ka nui ʻaʻole i ʻokoʻa nui ma waena o nā ʻano laʻana (p-value > 0.05). Hōʻike nā kiko kala i nā ʻokoʻa koʻikoʻi i ka nui (p-value ≤ 0.05). Hōʻike ʻia nā genes 20 me nā ʻokoʻa nui loa i ka nui i ka ʻulaʻula a me ka uliuli māmā (nā laʻana kaohi a me PPA), kēlā me kēia. ʻO ka nui o nā kiko melemele a me ka poni ma ka liʻiliʻi he 2.7 mau manawa ʻoi aku ka nui ma nā laʻana kaohi a me PPA ma mua o nā laʻana kaohi. Hōʻike nā kiko ʻeleʻele i nā genes me nā nui like ʻole, me nā ʻokoʻa CLR awelika ma waena o -1 a me 1. Ua helu ʻia nā waiwai P me ka hoʻohana ʻana i ka hoʻāʻo Mann-Whitney U a hoʻoponopono ʻia no nā hoʻohālikelike he nui me ka hoʻohana ʻana i ke kaʻina hana Benjamini-Hochberg. Pili nā genes i nā genes ʻelele ma ka papa inoa gene non-redundant. Aia nā inoa gene i ka hōʻailona KEGG e hōʻike ana i kahi gene KO. Hōʻike nā ʻokoʻa CLR awelika wiwo ʻole i nā nui like ʻole. Hōʻike kahi kaha pōkole (-) ʻaʻohe hōʻailona no ka gene i loko o ka waihona ʻikepili KEGG.
Ua ʻike ʻia nā taxa me nā genes e pili ana i ka metabolism a me/a i ʻole ka hana ʻana o ka PPA ma ka hoʻohālikelike ʻana i ka ʻike taxonomic o nā contigs me ka contig ID o ka gene. Ma ka pae genus, ua ʻike ʻia he 130 genera e pili ana i ka metabolism PPA a ua ʻike ʻia he 61 genera e pili ana i ka hana ʻana o ka PPA (Papa Hoʻohui 4). Eia naʻe, ʻaʻohe genera i hōʻike i nā ʻokoʻa koʻikoʻi i ka nui (p > 0.05).
Ma ka pae ʻano, ua ʻike ʻia he 144 mau ʻano bacteria i loaʻa nā genes e pili ana i ka metabolism PPA a ua ʻike ʻia he 68 mau ʻano bacteria i loaʻa nā genes e pili ana i ka hana PPA (Papa Hoʻohui 5). Ma waena o nā metabolizers PPA, ʻewalu mau bacteria i hōʻike i ka piʻi nui ʻana o ka nui ma waena o nā ʻano laʻana, a ua hōʻike nā mea a pau i nā loli koʻikoʻi i ka hopena (Papa Hoʻohui 6). ʻO nā metabolizers PPA a pau i ʻike ʻia me nā ʻokoʻa koʻikoʻi i ka nui ua ʻoi aku ka nui ma nā laʻana PPA. Ua hōʻike ka hoʻokaʻawale ʻana o ka pae ʻano i nā ʻelele o nā genera i ʻokoʻa ʻole ma waena o nā ʻano laʻana, me kekahi mau ʻano Bacteroides a me Ruminococcus, a me Duncania dubois, Myxobacterium enterica, Monococcus pectinolyticus, a me Alcaligenes polymorpha. Ma waena o nā bacteria hana PPA, ʻehā mau bacteria i hōʻike i nā ʻokoʻa koʻikoʻi i ka nui ma waena o nā ʻano laʻana. ʻO nā ʻano me nā ʻokoʻa koʻikoʻi i ka nui, ʻo ia hoʻi ʻo Bacteroides novorossi, Duncania dubois, Myxobacterium enteritidis, a me Ruminococcus bovis.
Ma kēia haʻawina, ua nānā mākou i nā hopena o ka hōʻike ʻana o ka PPA ma ka microbiota ʻōpū o nā ʻiole. Hiki i ka PPA ke hoʻopuka i nā pane like ʻole i loko o ka bacteria no ka mea ua hana ʻia e kekahi mau ʻano, hoʻohana ʻia ma ke ʻano he kumu meaʻai e nā ʻano ʻē aʻe, a i ʻole he hopena antimicrobial. No laila, ʻo kona hoʻohui ʻana i ke kaiapuni ʻōpū ma o ka hoʻopihapiha ʻana i ka meaʻai e loaʻa paha nā hopena like ʻole ma muli o ka hoʻomanawanui, ka hiki ke hoʻopilikia, a me ka hiki ke hoʻohana iā ia ma ke ʻano he kumu meaʻai. Hiki ke hoʻopau ʻia nā ʻano bacteria koʻikoʻi a pani ʻia e nā mea i ʻoi aku ka pale ʻana i ka PPA a i ʻole hiki ke hoʻohana iā ia ma ke ʻano he kumu meaʻai, e alakaʻi ana i nā loli i ke ʻano o ka microbiota ʻōpū. Ua hōʻike kā mākou mau hopena i nā ʻokoʻa koʻikoʻi i ka haku mele microbial akā ʻaʻohe hopena i ka ʻokoʻa microbial holoʻokoʻa. Ua ʻike ʻia nā hopena nui loa ma ka pae ʻano, me ka ʻoi aku o 70 taxa i ʻokoʻa nui i ka nui ma waena o PPA a me nā laʻana kaohi (Papa Hoʻohui 2). Ua hōʻike ka loiloi hou ʻana o ka haku mele o nā laʻana i hōʻike ʻia e PPA i ka heterogeneity nui o nā ʻano microbial i hoʻohālikelike ʻia me nā laʻana i hōʻike ʻole ʻia, e hōʻike ana e hiki i ka PPA ke hoʻonui i nā ʻano ulu bacteria a kaupalena i nā heluna bacteria e hiki ke ola i nā kaiapuni waiwai PPA. No laila, hiki i ka PPA ke hoʻoulu koho i nā loli ma mua o ka hoʻopilikia nui ʻana i ka ʻokoʻa microbiota ʻōpū.
Ua hōʻike mua ʻia nā mea mālama meaʻai e like me ka PPA e hoʻololi i ka nui o nā ʻāpana microbiome o ka ʻōpū me ka ʻole o ka hoʻopilikia ʻana i ka ʻokoʻa holoʻokoʻa (Nagpal et al., 2021). Maanei, ua ʻike mākou i nā ʻokoʻa koʻikoʻi loa ma waena o nā ʻano Bacteroidetes i loko o ka phylum Bacteroidetes (i ʻike mua ʻia ʻo Bacteroidetes), kahi i hoʻonui nui ʻia i nā ʻiole i hōʻike ʻia i ka PPA. ʻO ka hoʻonui ʻia ʻana o ka nui o nā ʻano Bacteroides e pili ana me ka hoʻonui ʻia ʻana o ka mucus degradation, kahi e hoʻonui ai i ka pilikia o ka maʻi a hoʻoulu i ka mumū (Cornick et al., 2015; Desai et al., 2016; Penzol et al., 2019). Ua ʻike ʻia kekahi noiʻi ua hōʻike nā ʻiole kāne neonatal i mālama ʻia me Bacteroides fragilis i nā ʻano pilikanaka e hoʻomanaʻo ana i ka maʻi autism spectrum (ASD) (Carmel et al., 2023), a ua hōʻike nā noiʻi ʻē aʻe e hiki i nā ʻano Bacteroides ke hoʻololi i ka hana pale a alakaʻi i ka cardiomyopathy inflammatory autoimmune (Gil-Cruz et al., 2019). Ua hoʻonui nui ʻia nā ʻano o ka genera Ruminococcus, Prevotella, a me Parabacteroides i nā ʻiole i hōʻike ʻia i ka PPA (Coretti et al., 2018). Pili kekahi mau ʻano Ruminococcus me nā maʻi e like me ka maʻi Crohn ma o ka hana ʻana o nā cytokines proinflammatory (Henke et al., 2019), ʻoiai ʻo nā ʻano Prevotella e like me Prevotella humani e pili ana me nā maʻi metabolic e like me ke koko kiʻekiʻe a me ka insulin sensitivity (Pedersen et al., 2016; Li et al., 2017). ʻO ka mea hope loa, ua ʻike mākou ua haʻahaʻa loa ka lakio o Bacteroidetes (i ʻike mua ʻia ʻo Firmicutes) i Bacteroidetes i nā ʻiole i hōʻike ʻia i ka PPA ma mua o nā ʻiole kaohi ma muli o ka nui o nā ʻano Bacteroidetes. Ua hōʻike mua ʻia kēia lakio he hōʻailona koʻikoʻi o ka homeostasis ʻōpū, a ua pili nā haunaele i kēia lakio me nā ʻano maʻi like ʻole (Turpin et al., 2016; Takezawa et al., 2021; An et al., 2023), me nā maʻi mumū o ka ʻōpū (Stojanov et al., 2020). Ma ke ʻano hui, ʻike ʻia nā ʻano o ka phylum Bacteroidetes i hoʻopilikia nui ʻia e ka PPA meaʻai kiʻekiʻe. Hiki paha kēia ma muli o ka hoʻomanawanui kiʻekiʻe i ka PPA a i ʻole ka hiki ke hoʻohana i ka PPA ma ke ʻano he kumu ikehu, kahi i hōʻike ʻia he ʻoiaʻiʻo no ka liʻiliʻi o hoʻokahi ʻano, ʻo Hoylesella enocea (Hitch et al., 2022). Ma kekahi ʻaoʻao, hiki i ka hōʻike ʻana o ka PPA makuahine ke hoʻonui i ka ulu ʻana o ka fetal ma ka hoʻolilo ʻana i ka ʻōpū o nā keiki ʻiole i ʻoi aku ka maʻalahi i ka colonization Bacteroidetes; akā naʻe, ʻaʻole i ʻae kā mākou hoʻolālā noiʻi i kēlā loiloi.
Ua hōʻike ka loiloi ʻike metagenomic i nā ʻokoʻa koʻikoʻi i ka nui o nā genes e pili ana i ka metabolism PPA a me ka hana ʻana, me nā ʻiole i hōʻike ʻia e PPA e hōʻike ana i ka nui o nā genes kuleana no ka hana ʻana o PPA, ʻoiai ʻo nā ʻiole i hōʻike ʻole ʻia e PPA e hōʻike ana i ka nui o nā genes kuleana no ka metabolism PAA (Kiʻi 6). Hōʻike kēia mau hopena ʻaʻole paha ka hopena o PPA ma ka hoʻohui microbial ma muli wale nō o kona hoʻohana ʻana, inā ʻaʻole, ʻo ka nui o nā genes e pili ana i ka metabolism PPA e hōʻike i ka nui o ka nui i loko o ka microbiome ʻōpū o nā ʻiole i hōʻike ʻia e PPA. ʻO kekahi wehewehe ʻana, ʻo ia ka PPA e hoʻoponopono i ka nui o ka bacteria ma o kona mau hopena antimicrobial ma mua o kona hoʻohana ʻana e ka bacteria ma ke ʻano he meaʻai. Ua hōʻike nā haʻawina mua e kāohi ana ka PPA i ka ulu ʻana o Salmonella Typhimurium ma ke ʻano hilinaʻi i ka dose (Jacobson et al., 2018). ʻO ka hōʻike ʻana i nā ʻano kiʻekiʻe o ka PPA e koho paha no nā bacteria i kūʻē i kona mau waiwai antimicrobial a ʻaʻole paha e hiki ke metabolize a hana paha. No ka laʻana, ua hōʻike kekahi mau ʻano Parabacteroides i ka nui o ka nui i nā laʻana PPA, akā ʻaʻohe genes e pili ana i ka metabolism PPA a i ʻole ka hana ʻana i ʻike ʻia (Nā Papa Hoʻohui 2, 4, a me 5). Eia kekahi, ua hoʻolaha nui ʻia ka hana ʻana o PPA ma ke ʻano he huahana fermentation ma waena o nā bacteria like ʻole (Gonzalez-Garcia et al., 2017). ʻO ke ʻano like ʻole o ka bacteria ke kumu paha no ka nui o nā genes e pili ana i ka metabolism PPA i nā laʻana kaohi (Averina et al., 2020). Eia kekahi, he 27 wale nō (2.14%) o 1332 mau genes i wānana ʻia he mau genes e pili wale ana me ka metabolism PPA. He nui nā genes e pili ana me ka metabolism PPA e pili pū ana i nā ala metabolic ʻē aʻe. Hōʻike hou kēia i ka nui o nā genes i komo i ka metabolism PPA i nā laʻana kaohi; hiki i kēia mau genes ke hana ma nā ala i ʻole e hopena i ka hoʻohana ʻana a i ʻole ka hoʻokumu ʻana o PPA ma ke ʻano he huahana. I kēia hihia, hoʻokahi wale nō gene e pili ana me ka hanauna PPA i hōʻike i nā ʻokoʻa koʻikoʻi i ka nui ma waena o nā ʻano laʻana. I ka hoʻohālikelike ʻana i nā genes e pili ana i ka metabolism PPA, ua koho ʻia nā genes marker no ka hana ʻana o PPA no ka mea pili pono lākou i ke ala bacteria no ka hana ʻana o PPA. I loko o nā ʻiole i hōʻike ʻia e PPA, ua ʻike ʻia nā ʻano āpau i hoʻonui nui ʻia ka nui a me ka hiki ke hana i ka PPA. Kākoʻo kēia i ka wānana e koho nā PPA i nā mea hana PPA a no laila e wānana e hoʻonui ʻia ka hiki ke hana PPA. Eia nō naʻe, ʻaʻole pili pono ka nui o nā genes me ka hōʻike ʻana o nā genes; no laila, ʻoiai ʻoi aku ka nui o nā genes e pili ana me ka metabolism PPA i nā laʻana kaohi, ʻokoʻa paha ka nui o ka hōʻike ʻana (Shi et al., 2014). No ka hōʻoia ʻana i ka pilina ma waena o ka laha ʻana o nā genes e hana ana i ka PPA a me ka hana ʻana o PPA, pono nā haʻawina e pili ana i ka hōʻike ʻana o nā genes i komo i ka hana ʻana o PPA.
Ua hōʻike ka hoʻākāka hana o ka PPA a me nā metagenomes kaohi i kekahi mau ʻokoʻa. Ua hōʻike ka loiloi PCA o ka ʻike gene i nā hui ʻokoʻa ma waena o ka PPA a me nā laʻana kaohi (Kiʻi 5). Ua hōʻike ka hui ʻana i loko o ka laʻana he ʻoi aku ka ʻokoʻa o ka ʻike gene kaohi, ʻoiai ua hui pū nā laʻana PPA. Ua like ka hui ʻana ma o ka ʻike gene me ka hui ʻana ma o ka haku mele ʻano. No laila, kūlike nā ʻokoʻa i ka nui o ke ala me nā loli i ka nui o nā ʻano kikoʻī a me nā ʻano i loko o lākou. I nā laʻana PPA, ʻelua mau ala me ka nui kiʻekiʻe e pili ana i ka metabolism kō aminosugar/nucleotide (ko:K21279) a me nā ala metabolism lipid he nui (ko:K00647, ko:K03801; Papa Hoʻohui 3). ʻIke ʻia nā genes e pili ana me ko:K21279 e pili ana me ka genus Bacteroides, kekahi o nā genera me ka helu kiʻekiʻe o nā ʻano i nā laʻana PPA. Hiki i kēia enzyme ke pale aku i ka pane pale ma ka hōʻike ʻana i nā polysaccharides capsular (Wang et al., 2008). Hiki paha i kēia ke kumu no ka hoʻonui ʻia ʻana o Bacteroidetes i ʻike ʻia i nā ʻiole i hōʻike ʻia e PPA. Hoʻopiha kēia i ka hoʻonui ʻia ʻana o ka synthesis waikawa momona i ʻike ʻia ma ka microbiome PPA. Hoʻohana nā bacteria i ke ala FASIIko:K00647 (fabB) e hana i nā waikawa momona, kahi e hoʻohuli ai i nā ala metabolic host (Yao lāua ʻo Rock, 2015; Johnson et al., 2020), a ʻo nā loli i ka metabolism lipid e pāʻani paha i kahi kuleana i ka neurodevelopment (Yu et al., 2020). ʻO kekahi ala e hōʻike ana i ka hoʻonui ʻia ʻana o ka nui i nā laʻana PPA ʻo ia ka biosynthesis hormone steroid (ko:K12343). Ke ulu nei nā hōʻike e pili ana i kahi pilina inverse ma waena o ka hiki o ka microbiota gut ke hoʻohuli i nā pae hormone a e hoʻohuli ʻia e nā hormones, i hiki i nā pae steroid kiʻekiʻe ke loaʻa nā hopena olakino ma lalo (Tetel et al., 2018).
ʻAʻole kēia haʻawina me nā palena a me nā noʻonoʻo ʻana. ʻO kahi ʻokoʻa koʻikoʻi, ʻaʻole mākou i hana i nā loiloi physiological o nā holoholona. No laila, ʻaʻole hiki ke hoʻoholo pololei inā pili nā loli i ka microbiome me kekahi maʻi. ʻO kekahi noʻonoʻo ʻana, ua hānai ʻia nā ʻiole i loko o kēia haʻawina i ka meaʻai like me ko lākou mau makuahine. Hiki i nā haʻawina e hiki mai ana ke hoʻoholo inā hoʻomaikaʻi ka hoʻololi ʻana mai kahi meaʻai momona PPA i kahi meaʻai ʻole PPA i kona hopena ma ka microbiome. ʻO kekahi palena o kā mākou haʻawina, e like me nā mea ʻē aʻe he nui, ʻo ia ka nui o ka laʻana i kaupalena ʻia. ʻOiai hiki ke huki ʻia nā hopena kūpono, ʻo ka nui o ka laʻana e hāʻawi i ka mana helu nui aʻe i ka wā e nānā ai i nā hopena. Makaʻala hoʻi mākou e pili ana i ka huki ʻana i nā hopena e pili ana i ka pilina ma waena o nā loli i ka microbiome ʻōpū a me kekahi maʻi (Yap et al., 2021). ʻO nā mea hoʻohuihui e like me ka makahiki, ke kāne, a me ka ʻai hiki ke hoʻololi nui i ka haku mele ʻana o nā microorganism. Hiki i kēia mau mea ke wehewehe i nā kūlike ʻole i ʻike ʻia ma ka palapala e pili ana i ka pilina o ka microbiome ʻōpū me nā maʻi paʻakikī (Johnson et al., 2019; Lagod lāua ʻo Naser, 2023). No ka laʻana, ua hōʻike ʻia nā lālā o ka genus Bacteroidetes ua hoʻonui ʻia a emi paha i nā holoholona a me nā kānaka me ASD (Angelis et al., 2013; Kushak et al., 2017). Pēlā nō, ua ʻike nā haʻawina o ka haku mele ʻana o ka ʻōpū i nā poʻe maʻi me nā maʻi ʻōpū mumū i ka hoʻonui a me ka emi ʻana i ka taxa like (Walters et al., 2014; Forbes et al., 2018; Upadhyay et al., 2023). No ka palena ʻana i ka hopena o ka manaʻo hoʻokae kāne, ua hoʻāʻo mākou e hōʻoia i ka hōʻike like ʻana o nā kāne i mea e hoʻokele ʻia ai nā ʻokoʻa e ka ʻai. ʻO kahi pilikia o ka annotation hana ʻo ia ka wehe ʻana i nā moʻo gene redundant. Pono kā mākou ʻano hana clustering gene i ka 95% ʻike moʻo a me ka 85% like o ka lōʻihi, a me ka uhi alignment 90% e hoʻopau i ka clustering wahaheʻe. Eia nō naʻe, i kekahi mau hihia, ua ʻike mākou i nā COG me nā annotation like (e.g., MUT) (Kiʻi 6). Pono nā haʻawina hou aʻe e hoʻoholo ai inā he ʻokoʻa kēia mau orthologs, e pili ana i nā genera kikoʻī, a i ʻole he palena kēia o ke ʻano hoʻohuihui gene. ʻO kekahi palena o ka annotation hana ʻo ia ka hewa kūpono; ʻo ka bacterial gene mmdA kahi enzyme i ʻike ʻia e pili ana i ka synthesis propionate, akā ʻaʻole pili ʻo KEGG iā ia me ke ala metabolic propionate. I ka hoʻohālikelike ʻana, pili nā scpB a me mmcD orthologs. ʻO ka nui o nā genes me ka ʻole o nā knockouts i koho ʻia e hopena i ka hiki ʻole ke ʻike i nā genes e pili ana i ka PPA i ka wā e loiloi ai i ka nui o ka gene. E pōmaikaʻi nā haʻawina e hiki mai ana mai ka nānā ʻana o ka metatranscriptome, hiki ke hāʻawi i kahi ʻike hohonu o nā ʻano hana o ka microbiota gut a hoʻopili i ka hōʻike ʻana o ka gene i nā hopena hiki ke iho. No nā haʻawina e pili ana i nā maʻi neurodevelopmental kikoʻī a i ʻole nā ​​​​maʻi ʻōpū mumū, pono nā loiloi physiological a me nā ʻano o nā holoholona e hoʻopili i nā loli i ka haku mele microbiome i kēia mau maʻi. ʻO nā haʻawina hou e hoʻololi ana i ka microbiome gut i loko o nā ʻiole germ-free e pono nō hoʻi e hoʻoholo inā he mea hoʻokele a ʻano paha ka microbiome o ka maʻi.
I ka hōʻuluʻulu manaʻo, ua hōʻike mākou he mea nui ka PPA meaʻai i ka hoʻololi ʻana i ke ʻano o ka microbiota ʻōpū. ʻO PPA kahi mea mālama i ʻae ʻia e ka FDA i loaʻa nui ʻia i nā meaʻai like ʻole, ma hope o ka hoʻohana lōʻihi ʻana, hiki ke alakaʻi i ka hoʻopilikia ʻana i ka flora ʻōpū maʻamau. Ua ʻike mākou i nā loli i ka nui o kekahi mau bacteria, e hōʻike ana e hiki i ka PPA ke hoʻololi i ke ʻano o ka microbiota ʻōpū. Hiki i nā loli i ka microbiota ke alakaʻi i nā loli i nā pae o kekahi mau ala metabolic, hiki ke alakaʻi i nā loli physiological e pili ana i ke olakino o ka mea hoʻokipa. Pono nā haʻawina hou aʻe e hoʻoholo ai inā hiki i nā hopena o ka PPA meaʻai ma ke ʻano microbial ke alakaʻi i ka dysbiosis a i ʻole nā ​​​​maʻi ʻē aʻe. Hoʻokumu kēia haʻawina i ke kumu no nā haʻawina e hiki mai ana i ka hopena o nā hopena PPA ma ke ʻano o ka ʻōpū i ke olakino kanaka.
Loaʻa nā ʻikepili i hōʻike ʻia ma kēia haʻawina ma nā waihona pūnaewele. ʻO ka inoa waihona a me ka helu komo: https://www.ncbi.nlm.nih.gov/, PRJNA1092431.
Ua ʻāpono ʻia kēia haʻawina holoholona e ke Komite Mālama a me ka Hoʻohana Holoholona o ke Kulanui o Central Florida (UCF-IACUC) (Helu ʻAe Hoʻohana Holoholona: PROTO202000002). Kūlike kēia haʻawina me nā kānāwai kūloko, nā lula, a me nā koi o ka ʻoihana.
NG: Manaʻo, Mālama ʻikepili, Loiloi kūhelu, Noiʻi, ʻAno Hana, Polokalamu, Nānā ʻIke, Kākau (palapala kumu), Kākau (loiloi & hoʻoponopono). LA: Manaʻo, Mālama ʻikepili, ʻAno Hana, Nā Kumuwaiwai, Kākau (loiloi & hoʻoponopono). SH: Loiloi kūhelu, Polokalamu, Kākau (loiloi & hoʻoponopono). SA: Noiʻi, Kākau (loiloi & hoʻoponopono). Luna kānāwai nui: Noiʻi, Kākau (loiloi & hoʻoponopono). SN: Manaʻo, Hoʻokele papahana, Nā Kumuwaiwai, Nānā, Kākau (loiloi & hoʻoponopono). TA: Manaʻo, Hoʻokele papahana, Nānā, Kākau (loiloi & hoʻoponopono).
Ua haʻi aku nā mea kākau ʻaʻole lākou i loaʻa ke kākoʻo kālā no ka noiʻi, ka mea kākau, a/a i ʻole ka paʻi ʻia ʻana o kēia ʻatikala.
Ke hōʻike nei nā mea kākau ua hana ʻia ka noiʻi me ka loaʻa ʻole o kekahi pilina kālepa a kālā paha i hiki ke wehewehe ʻia he paio hoihoi. ʻaʻole pili.
ʻO nā manaʻo a pau i hōʻike ʻia ma kēia ʻatikala, ʻo ia wale nō nā manaʻo o nā mea kākau a ʻaʻole pono e hōʻike i nā manaʻo o kā lākou mau ʻoihana, nā mea hoʻopuka, nā mea hoʻoponopono, a i ʻole nā ​​mea loiloi. ʻO nā huahana i loiloi ʻia ma kēia ʻatikala, a i ʻole nā ​​​​koi a kā lākou mea hana, ʻaʻole i hōʻoia ʻia a ʻae ʻia paha e ka mea hoʻopuka.
Hiki ke loaʻa nā mea kōkua no kēia ʻatikala ma ka pūnaewele: https://www.frontiersin.org/articles/10.3389/frmbi.2024.1451735/full#supplementary-material
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