Propionic acid (PPA), rungrul thahnak sii le rawl ah hmanmi thilri, nih hin vok ah a ttha lomi thluak tthanchonak a chuahter i cu he cun pawpi riantuan ningcang lo a chuahter, cucu pawfah riantuan ningcang lo ruangah a si kho. Rawl eidin PPA hmuhnak le pawpi chung thilnung hme tete a rawkralnak karlak ah pehtlaihnak a um tiah ruahnak an pek, asinain direct in hlathlainak tuah a si rih lo. Hika ah, dysbiosis a chuahter khomi gut microbiota fonhnak ah PPA he aa pehtlaimi thlennak hna kha kan hlathlai. Thlopbul lomi rawl (n=9) le PPA-a tammi rawl (n=13) pekmi vok hna i an paw chung microbiomes cu microbial composition le bacteria metabolic lamthluan aa dannak tuaktan awkah long-range metagenomic sequencing hmangin an i tthen. Rawl eidin PPA cu Bacteroides, Prevotella, le Ruminococcus phun zeimawzat telh in a biapi mi taxa tamnak he pehtlaihnak a ngei, cu hna chungtel hna cu a hlan ah PPA chuahternak ah an rak i tel cang. PPA-a tongmi vok hna i an microbiomes zong nih lipid metabolism le steroid hormone biosynthesis he aa pehtlaimi lam tam deuh an ngei. Kan hmuhmi nih PPA nih pawpi chung thilnung hme tete le aa pehtlaimi thilri thlennak lam kha a thlen khawh ti a langhter. Hi hmuhmi thlennak hna nih hin ei awk ah a him tiah thleidanmi thilri kilvengtu hna nih hin pawpi chung thilnung hme tete hna an i fonh ning le, cu nih cun minung ngandamnak zong a thlen khawh ti kha a langhter. Cu hna lakah, P, G asiloah S cu hlathlainak tuahmi thleidannak tlukruannak cungah aa hngat in thim a si. A hmaan lomi a ttha mi thleidannak nih a chuahpimi thil kha tlawmter awkah, a tlawmbik 1e-4 (1/10,000 relmi) tlukruannak tlukruannak kha hman a si. Statistical hlathlainak tuah hlan ah, Bracken nih a langhtermi aa pehtlaimi tamnak (fraction_total_reads) cu centered log-ratio (CLR) thlennak (Aitchison, 1982) hmangin thlen an si. CLR phunglam cu data thlennak caah thim a si, zeicahtiah a tahfung aa thleng lomi a si i a tlawm lomi dataset caah a za (Gloor et al., 2017). CLR thlennak nih a chuakmi logarithm a hman. Bracken nih a thanhmi relnak data cu aa pehtlaimi log langhternak (RLE) hmangin a tlarimi ah an ser (Anders le Huber, 2010). Matplotlib v. 3.7.1, seaborn v. 3.7.2 le sequential logarithms (Gloor et al., 2017) fonh in hmanthlak pawl cu an ser. 0.12.2 le stantanotations v. 0.5.0 (Hunter, 2007; Waskom, 2021; Charlier et al., 2022). Bacillus/Bacteroidetes ratio cu sample pakhat cio caah a tlarimi bacteria relnak hmangin tuak a si. Cabuai chung i langhtermi man hna cu decimal 4 tiang an i tthen. Simpson diversity index cu KrakenTools v. 1.2 package (Lu et al., 2022) ah pekmi alpha_diversity.py script hmangin tuak a si. Bracken report cu script ah pek a si i Simpson index “Si” cu -an parameter caah pek a si. A tamnak ah a biapi mi thleidannak cu CLR thleidannak ≥ 1 asiloah ≤ -1 tiah fianternak an tuah. A tlangpi in CLR aa dannak ±1 nih sample phunkhat a tamnak ah 2.7-fold a karh ti a langhter. Hmelchunhnak (+/-) nih PPA sample le control sample ah taxon a tam deuh maw tam lo ti a langhter. A sullam cu Mann-Whitney U hneksaknak (Virtanen et al., 2020) hmangin tuak a si. Statsmodels v. 0.14 (Benjamini le Hochberg, 1995; Seabold le Perktold, 2010) kha hman a si, cun hneksaknak tampi remh awkah Benjamini-Hochberg phunglam kha hman a si. Remhmi p-value ≤ 0.05 cu zatlang sullam hngalh khawhnak caah hramthawknak ah hman a si.
Minung microbiome cu “pum chung thilri hmanung bik” tiah an auh tawn i minung ngandamnak ah a biapi tukmi rian a ttuan (Baquero le Nombela, 2012). A bik in, pawpi microbiome cu a herhmi rian tampi ah a riantuannak le a riantuannak caah theih a si. Commensal bacteria cu pawpi chungah an tam, pawngkam hmun tampi an lak, rawl an hman, cun zawtnak hrik a chuahter khomi he an i zuam (Jandhyala et al., 2015). Gut microbiota chung i a phunphun in a ummi bacteria hna nih vitamin tibantuk a herhmi rawl an chuah khawh i rawl ei khawhnak an bawmh khawh (Rowland et al., 2018). Bacterial metabolites zong nih tissue tthanchonak a thlen i metabolic le immune lam a tthanchoter ti zong hmuh a si (Heijtz et al., 2011; Yu et al., 2022). Minung pawpi chung i a ummi thilnung hme tete hna i an i fonh ning cu a phunphun in a um i, rawl eidin, nu le pa sinak, sii, le ngandamnak dirhmun tibantuk hrinsor le pawngkam thil sining cungah aa hngat (Kumbhare et al., 2019).
Nu rawl cu nau le ngakchia tthanchonak ah a biapi tukmi a si i tthanchonak a thlen khomi thilri hna a chuahtertu a si tiah ruahmi a si (Bazer et al., 2004; Innis, 2014). Cu bantuk thilri pakhat cu propionic acid (PPA) a si, cu cu rungrul cawhnak in hmuhmi a tawimi thau acid a si i rawl ah aa telmi a si (den Besten et al., 2013). PPA nih hin rungrul thahnak le rungrul thahnak a ngeih caah rawl kilvennak le riantuannak ah hmanmi a si i rungrul le rungrul karhternak khamnak caah hman a si (Wemmenhove et al., 2016). PPA nih hin tissue dangdang ah thlennak a chuahpi. Thin chung ah, PPA nih macrophages chung i cytokine langhternak a hnorsuannak thawngin fahnak a kham khawh (Kawasoe et al., 2022). Hi uknak tthatnak cu a dang ralvennak cells zongah hmuh a si, cu nih cun fahnak a zorter (Haase et al., 2021). Sihmanhsehlaw, thluak ah a kalhmi thil sining hmuh a si. Hlan lio hlathlainak nih PPA hmuhnak nih vok ah autism bantuk ziaza a chuahter ti a langhter (El-Ansary et al., 2012). Hlathlainak dang nih PPA nih thluak chung i gliosis a chuahter khawh i thluak chung i fahnak lam a chuahter khawh ti an langhter (Abdelli et al., 2019). PPA cu a derthawmmi acid a si caah, pawpi chung epithelium in thisen chungah a karh kho i cucaah thisen-thluak khamnak le nauinn (Stinson et al., 2019) telh in khamnak khamnak kha a tan khawh (Stinson et al., 2019), PPA cu bacteria nih a chuahtermi uknak thilri pakhat a si ti a biapitnak kha a langhter. PPA nih autism zawtnak caah tihnung a si khomi rian cu hlathlainak tuah cuahmah lio a si ko nain, autism zawtnak a ngeimi hna cungah a chuahpimi thil cu thluak thleidannak chuahternak leng tiang a phan kho.
Pawpi zawtnak hmelchunhnak hna cu zunthlum le zunthlum tibantuk hna hi thluak tthanchonak lei zawtnak a ngeimi mizaw hna ah an um tawn (Cao et al., 2021). Hlan lio hlathlainak nih a langhter ning ahcun autism spectrum zawtnak (ASD) a ngeimi mizaw hna i an microbiome cu a ngandammi hna he aa dang, cu nih cun gut microbiota dysbiosis a um ti a langhter (Finegold et al., 2010). Cu bantuk cun, zunthlum zawtnak, thau tuk, Alzheimer zawtnak, tbk. a ngeimi mizaw hna i an microbiome sining zong a ngandammi hna he aa dang (Turnbaugh et al., 2009; Vogt et al., 2017; Henke et al., 2019). Sihmanhsehlaw, nihin tiangah, pawpi microbiome le thluak lei zawtnak asiloah zawtnak hmelchunhnak karlak ah a ruang pehtlaihnak a um lo (Yap et al., 2021), asinain hi zawtnak dirhmun cheukhat ah hin bacteria phun zeimawzat nih hmunhma an lak tiah ruah a si. Tahchunhnak ah, Akkermansia, Bacteroides, Clostridium, Lactobacillus, Desulfovibrio le a dang phun hna cu autism zawtnak a ngeimi hna i an microbiota ah an tam deuh (Tomova et al., 2015; Golubeva et al., 2017; Cristiano et al., 2018; Zurita et al., 2020). A hlei in, hi phun cheukhat chungtel phun hna cu PPA chuahternak he aa pehtlaimi genes an ngei ti theih a si (Reichardt et al., 2014; Yun le Lee, 2016; Zhang et al., 2019; Baur le Dürre, 2023). PPA nih a ngeihmi rungrul thah khawhnak a ngeih caah, a tamnak karhternak nih PPA a chuahtertu rungrul karhternak caah thathnemnak a chuahpi kho (Jacobson et al., 2018). Cucaah, PFA a tammi pawngkam nih cun pawpi chung zawtnak rungrul telh in pawpi chung thilnung hme tete ah thlennak a chuahter khawh, cucu pawpi chung zawtnak hmelchunhnak a chuahter khomi thil a si kho.
Microbiome hlathlainak ah a biapi bikmi biahalnak cu microbial fonhmi aa dannak cu a tanglei zawtnak a chuahtertu asiloah hmelchunhnak a si maw ti hi a si. Rawl eidin, pawpi chung thilnung hme tete le thluak lei zawtnak karlak i a harmi pehtlaihnak fianternak caah a hmasa bik kehlan cu rawl eidin nih thilnung hmete sining cung ah a chuahpimi thil kha tuaktan ding a si. Cu caah, PPA-a tammi asiloah PPA-a tlawmmi rawl pekmi vok tefa hna i an pawpi microbiomes tahchunh awkah caan saupi relmi metagenomic sequencing kan hman. A fale cu an nu hna he aa khatmi rawl an pek hna. PPA a tammi rawl ei nih hin pawpi chung i a ummi thilnung hme tete fonhmi le thilnung hme tete riantuannak lam ah thlennak a chuahpi lai tiah kan ruah, a bik in PPA thlennak le/asiloah PPA chuahternak he aa pehtlaimi hna kha.
Hi hlathlainak nih hin FVB/N-Tg(GFAP-GFP)14Mes/J transgenic mice (Jackson Laboratories) kha a hman hna i, cu nih cun glia-specific GFAP promoter uknak tangah green fluorescent protein (GFP) kha a tam tuk in a langhter i, University of Central Florida Institutional Animal Care and Use Committee (UCIA-CU) nawlpeknak nambar: PROTO202000002). Nu le pa hnuk dinh hnu ah, vok pawl cu vok pakhat cio ah nu le pa 1-5 cio in chiah an si. Zu pawl cu an duh paoh in thianh cangmi uknak rawl (thlenmi open-label standard diet, 16 kcal% thau) asiloah sodium propionate-chapmi rawl (thlenmi open-label standard diet, 16 kcal% thau, 5,000 ppm sodium propionate aa telmi) in ad libitum in an pek hna. Sodium propionate hmanmi zat cu rawl rit dihlak 5,000 mg PFA/kg he aa tluk. Mah cu rawl kilvennak caah hman ding in cohlanmi PPA a tam bik a si. Hi hlathlainak caah timhtuahnak ngeih awkah, hringtu vok pawl cu an i umh hlan zarh 4 chung rawl pahnih an pek hna i dam nau a pawi chung vialte an pehzulh hna. A chuakmi vok [vok 22, control 9 (pa 6, nu 3) le PPA 13 (pa 4, nu 9)] cu hnuk an dinh hna i thla 5 chung dam pawl he rawl an ei ning tein an pehzulh hna. Thla 5 an si ah fale vok pawl cu raithawinak ah an thah hna i an pawpi chung zunthlum pawl cu an lak hna i a hramthawk ah 1.5 ml microcentrifuge tubes ah -20°C ah an chiah hna i cu hnu ah -80°C freezer ah an chiah hna i host DNA a dih tiang le microbial nucleic acids an chuah hlan tiang an chiah hna.
A ngeitu DNA cu remhmi phunglam ningin lak a si (Charalampous et al., 2019). A tawinak in, zunthlum chung thilri pawl cu 500 μl InhibitEX (Qiagen, Cat#/ID: 19593) ah an thlen hna i a linmi ah an chiah hna. Chuahnak pakhat ah a tam bik zunthlum 1-2 tiang tuah. Cu hnuah zunthlum chung thilri pawl cu hri chung i plastic hriamnam hmangin hriamnam in an i fonh i zunthlum an ser. Sample pawl kha 10,000 RCF ah 5 min chung asiloah sample pawl an i tthen hlan tiang centrifuge tuah, cun a cunglei ti kha thlet law 250 μl 1× PBS ah pellet kha hnim ṭhan. Eukaryotic cell vun hna kha tthawnternak caah 250 μl 4.4% saponin cawhnuk (TCI, thilri nambar S0019) kha sample ah cawh. Sample pawl cu a nem tiang nem tein cawh an si i innchung lumnak ah minutes 10 chung an chiah hna. A hnu ah, eukaryotic cells hrawh awkah, 350 μl nuclease-free ti kha sample ah an cawh, 30 s chung an chiah, cun 12 μl 5 M NaCl an cawh. Cu hnu ah sample pawl cu 6000 RCF ah minutes 5 chung an thlet hna. A cunglei ti kha thlet law 100 μl 1X PBS ah pellet kha hnim ṭhan. Host DNA chuah awkah, 100 μl HL-SAN buffer (12.8568 g NaCl, 4 ml 1M MgCl2, 36 ml nuclease-free water) le 10 μl HL-SAN enzyme (ArticZymes P/N 70910-202) kha cawh. Samples pawl cu pipetting in ttha tein an cawh hna i 37 °C ah 30 min chung 800 rpm ah EppendorfTM ThermoMixer C ah an chiah hna. An chiah hnu ah, 6000 RCF ah 3 min chung an thlet hna i 800 μl le 1000 μl PX1 PBS in voihnih an tawl hna. A donghnak ah, 100 μl 1X PBS ah pellet kha hnim ṭhan.
Bacteria DNA dihlak cu New England Biolabs Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, Cat# T3010L) hmangin thleidan a si. Kit he aa pehtlaimi riantuan ning phunglam cu tlawmpal remh a si. A donghnak thletnak caah riantuan hlan ah nuclease aa tel lomi ti kha 60°C ah chiah law chiah. Sample pakhat ah 10 μl Proteinase K le 3 μl RNase A chap. Cun 100 μl Cell Lysis Buffer cawh law nem tein cawh. Cu hnu ah zohchunhmi pawl cu EppendorfTM ThermoMixer C ah 56°C le 1400 rpm ah a tlawmbik suimilam 1 le suimilam 3 tiang an chiah hna. Incubated samples cu 12,000 RCF ah minutes 3 chung centrifuge an tuah hna i sample pakhat cio chungin a chuakmi ti cu 400 μL binding solution aa telmi a dang 1.5 mL microcentrifuge tube ah an thlen. Cu hnu ah hri pawl cu second 1 karlak ah second 5-10 chung pulse vortex an tuah hna. Sample pakhat cio chung i a ummi ti vialte (600-700 μL hrawng) kha a luangmi laknak hri chungah chiahmi filter cartridge ah thlen. A hramthawk DNA pehtlaihnak tuah khawhnak ding caah tube pawl cu 1,000 RCF ah minutes 3 chung an thlet hna i a tangmi ti chuah khawhnak ding caah 12,000 RCF ah minutes 1 chung an thlet hna. Sample column cu laknak hri thar ah thlen a si i voihnih tawl a si. A voikhatnak tawlnak caah, 500 μL tawlnak buffer kha tube pakhat ah cawh. Tube kha voi 3-5 tiang thlen law 12,000 RCF ah minutes 1 chung centrifuge tuah. Ti kha laknak hri chungin hlonh law filter cartridge kha laknak hri chung lila ah chiah ṭhan. A voihnihnak tawlnak caah, 500 μL tawlnak buffer kha filter ah thlen loin cawh. Samples cu 12,000 RCF ah minutes 1 chung an thlet hna. Filter cu 1.5 mL LoBind® tube ah thlen law 100 μL a lum cangmi nuclease-free ti kha cawh. Filter pawl cu innchung lumnak ah minutes 1 chung an chiah hna i minutes 1 chung 12,000 RCF ah an thlet hna. A chuakmi DNA cu -80°C ah chiah a si.
DNA tlawmtam cu QubitTM 4.0 Fluorometer hmangin tah a si. DNA cu sertu nih a chimmi ningin QubitTM 1X dsDNA High Sensitivity Kit (Cat. No. Q33231) hmangin timhtuahnak a si. DNA tthencheunak sauhnak tthencheunak cu AglientTM 4150 asiloah 4200 TapeStation hmangin tah a si. DNA cu AgilentTM Genomic DNA Reagents (Cat. No. 5067-5366) le Genomic DNA ScreenTape (Cat. No. 5067-5365) hmangin timhtuahnak a si. Cauk chiahnak inn timhtuahnak cu Oxford Nanopore TechnologiesTM (ONT) Rapid PCR Barcoding Kit (SQK-RPB004) hmangin sertu lamhruainak ningin tuah a si. DNA cu ONT GridIONTM Mk1 sequencer hmangin Min106D flow cell (R 9.4.1) he sequence tuah a si. Sequencing settings cu: a dikthliar in a sangmi hrampi auhnak, a tlawmbik q man 9, barcode chiahnak, le barcode tlawmternak hna an si. Samples pawl cu suimilam 72 chung an i tthen, cu hnu ah base call data cu a dang riantuannak le hlathlainak caah an pek hna.
Bioinformatics riantuannak cu a hlan ah langhtermi lam hna hmangin tuah a si (Greenman et al., 2024). Sequencing in hmuhmi FASTQ file pawl cu sample pakhat cio caah directory ah an ṭhen hna. Bioinformatics hlathlainak tuah hlanah, data cu a tanglei pipeline hmangin an tuah: pakhatnak ah, sample pawl i FASTQ file pawl cu FASTQ file pakhat ah an fonh hna. Cun, 1000 bp nak tawi deuhmi relmi hna cu Filtlong v. 0.2.1 hmangin an thleidan hna, thlenmi parameter pakhat lawng a ummi cu –min_length 1000 (Wick, 2024) a si. A dang thleidan hlan ah, relnak tthatnak cu NanoPlot v. 1.41.3 hmangin a tanglei parameters he uk a si: –fastq –plots dot –N50 -o
Taxonomic thleidannak caah, relmi le fonhmi contigs cu Kraken2 v. 2.1.2 ( Wood et al., 2019 ) hmangin tthen an si. Relmi le fonhmi caah report le chuahmi file pawl kha pakhat cio in ser. Relmi le fonhmi hna hlathlainak caah –use-names thimnak kha hmang. –gzip-compressed le –paired thimnak hna cu relmi tthen caah langhter an si. Metagenomes chung i taxa a tamnak cu Bracken v. 2.8 ( Lu et al., 2017 ) hmangin tuak a si. A tanglei parameters he bracken-build hmangin base 1000 aa telmi kmer database kan ser hmasa: -d
Gene fianternak le aa pehtlaimi tamnak tuaktannak cu Maranga le a hawile nih an langhtermi phunglam remhmi hmangin tuah a si. (Maranga et al., 2023). Pakhatnak ah, 500 bp nak tawi deuhmi contigs cu SeqKit v. 2.5.1 (Shen et al., 2016) hmangin pumhnak vialte chungin an chuah hna. Thimmi pumhnak hna cu pan-metagenome ah an fonh hna. Open reading frames (ORFs) cu Prodigal v. 1.0.1 (Prodigal v. 2.6.3 he aa tlukmi version) hmangin a tanglei parameters hna he hngalh khawh an si: -d
Genes pawl cu Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO) identifiers ningin eggNOG nih gene lam tamnak tahchunhnak ah an chiah hna. Knockout a ngei lomi genes asiloah knockout tampi a ngeimi genes cu hlathlainak tuah hlanah an lak hna. Cu hnu ah sample pakhat ah KO pakhat cio i a tlangpi in a tamnak kha tuak a si i zatlang hlathlainak tuah a si. PPA metabolism genes cu KEGG_Pathway column ah row ko00640 pekmi gene paohpaoh tiah fianternak an tuah, KEGG ningin propionate metabolism ah rian a ttuanmi a langhter. PPA chuahternak he aa pehtlaimi tiah hngalhmi genes pawl cu Supplementary Table 1 (Reichardt et al., 2014; Yang et al., 2017) ah an langhter. Sample phunkhat cio ah a tam deuhmi PPA metabolism le chuahnak genes pawl hngalh khawhnak ding caah thlennak hneksaknak an tuah. Hlathlainak tuahmi gene pakhat cio caah thlennak thongkhat an tuah. Statistical significance hngalh khawhnak ding caah p-value 0.05 cu cutoff ah hman a si. Riantuannak fianternak hna cu phu chung i aiawhtu gene hna fianternak cungah hram bunh in phu pakhat chung i gene pakhat cio sinah pek an si. PPA metabolism le/asiloah PPA chuahnak he aa pehtlaimi taxa cu Kraken2 chuahmi file chung i contig IDs pawl kha eggNOG hmangin riantuannak fianternak tuah lio ah chiahmi contig IDs he aa tlakmi in hngalh khawh a si. A biapitnak hneksaknak cu a hlan ah langhtermi Mann-Whitney U hneksaknak hmangin tuah a si. Hneksaknak tampi caah remhnak cu Benjamini-Hochberg phunglam hmangin tuah a si. Statistical significance hngalh khawhnak ding caah p-value ≤ 0.05 cu cutoff ah hman a si.
Simpson phunphun index hmangin zunthlum hna i an pawpi chung thilnung hme tete aa dangmi kha an tuak. Control le PPA sample karlak ah phun le phun phunphun kongah thleidannak a um lo (phun caah p-man: 0.18, phun caah p-man: 0.16) (Figure 1). Cu hnu ah microbial fonhmi cu a hrampi thilri hlathlainak (PCA) hmangin tahchunh a si. Figure 2 nih an phyla ning in sample pawl an i fonhnak a langhter, PPA le control sample karlak ah microbiomes phun hna i an i dannak a um ti a langhter. Hi fonhmi cu phun tlukruannak ah a lang lo deuh, cu nih cun PPA nih bacteria cheukhat a hnorsuan hna ti a langhter (Supplementary Fig. 1).
Hmanthlak 1. Mouse pawpi chung i a ummi phun le phun hna i an alpha phunphun. PPA le control sample chung i phun (A) le phun (B) hna i Simpson phunphun tahfung a langhtermi box plots. A sullam cu Mann-Whitney U hneksaknak hmangin hngalh khawh a si, cun Benjamini-Hochberg phunglam hmangin remhnak tampi tuah a si. ns, p-value cu a biapi lo (p>0.05).
Hmanthlak 2. Saram phun cio ah vok paw chung microbiome fonhnak ah a biapi bikmi thilri hlathlainak a phichuak. A biapi bikmi thilri hlathlainak plot nih a hmasa bik a biapi bikmi thilri pahnih karlak ah sample pawl an i phawtzamhnak kha a langhter. Rong nih sample phun a langhter: PPA-a tongmi vok cu a senmi an si i control vok cu a senmi an si. A biapi bikmi thilri 1 le 2 cu x-axis le y-axis ah an suai hna i, an fiantermi aa dannak zat in an langhter.
RLE thlenmi relnak data hmangin, control le PPA mice (control: 9.66, PPA: 3.02; p-value = 0.0011) ah Bacteroidetes/Bacilli ratio karlak ah a langmi zornak hmuh a si. Hi aa dannak cu PPA zunthlum ah Bacteroidetes a tam deuh caah a si, asinain aa dannak cu a biapi lo (control mean CLR: 5.51, PPA mean CLR: 6.62; p-value = 0.054), cu lioah Bacteroidetes a tam ning cu aa lo (control mean CLR: 7.76, PPA-value: 0.066; = 0.18).
Gut microbiome chungtel hna an tamnak hlathlainak nih PPA le control sample karlak ah phylum 1 le phun 77 cu aa dang ngaingai ti a langhter (Supplementary Table 2). PPA sample chung i phun 59 an tamnak cu control sample nakin a tam deuh ngaingai, cun control sample ah phun 16 lawng an tamnak cu PPA sample nakin a tam deuh (Figure 3).
Figure 3. PPA le control mice hna i an pawpi microbiome chung i taxa an tamnak aa dannak. Meitlang hmanthlak nih PPA le control sample karlak ah phun (A) asiloah phun (B) tamnak ah aa dannak a langhter. A senmi dot nih taxa tamnak ah a biapi mi thleidannak a um lo ti a langhter. A rong aa dawhmi dot nih a tamnak ah a biapi mi thleidannak a langhter (p-value ≤ 0.05). Sample phun karlak ah a tam bik aa dannak a ngeimi a sangbik 20 taxa cu a sen le a sen (control le PPA sample) in langhter an si. A senmi le a senmi dot pawl cu control asiloah PPA sample ah control nakin a tlawmbik let 2.7 in an tam deuh. A senmi dot nih a tlawmtam aa dang tukmi taxa a aiawh, CLR aa dannak cu -1 le 1 karlak a si. P man cu Mann-Whitney U hneksaknak hmangin tuak a si i Benjamini-Hochberg phunglam hmangin hneksaknak tampi caah remh a si. Bold mean CLR aa dannak nih a tamnak ah a biapi mi aa dannak a langhter.
Pawpi chung i a ummi thilnung hme tete hna an i fonh ning kan hlathlai hnu ah, thilnung hmete hna i an riantuan ning fianternak kan tuah. A ttha lomi gene pawl kha an thleidan hna hnu ah, sample vialte ah aa dangmi gene 378,355 an hmuh hna. Hi genes pawl i an i thlengmi tamnak cu a hrampi thilri hlathlainak (PCA) caah hman a si, cun a phichuak nih an riantuannak sining cung ah hram bunh in sample phun pawl i fonhnak sangpi a langhter (Figure 4).
Hmanthlak 4. PCA hmuhmi cu vok pawpi microbiome riantuannak sining hmangin. PCA plot nih a hmasa bik a biapi bikmi thilri pahnih ah sample pawl an i phawtzamhnak kha a langhter. Rong nih sample phun a langhter: PPA-a tongmi vok cu a senmi an si i control vok cu a senmi an si. A biapi bikmi thilri 1 le 2 cu x-axis le y-axis ah an suai hna i, an fiantermi aa dannak zat in an langhter.
A hnu ah sample phun dangdang ah KEGG knockouts a tamnak kha kan zoh. A dihlak ah aa dangmi knockout 3648 an hmuh, cu chung ah 196 cu control sample ah a tam deuh i 106 cu PPA sample ah a tam deuh (Figure 5). Control sample ah a dihlak ah gene 145 le PPA sample ah gene 61 an hmuh, an tam ning aa dang ngai. PPA sample ah lipid le aminosugar thlennak he aa pehtlaimi lam hna cu a hlei in an rum deuh (Supplementary Table 3). Nitrogen thlennak le sulfur relay system he aa pehtlaimi lam hna cu control sample ah a tam deuh in an rum (Supplementary Table 3). Aminosugar/nucleotide metabolism (ko:K21279) le inositol phosphate metabolism (ko:K07291) he aa pehtlaimi genes tamnak cu PPA sample ah a tam deuh ngaingai (Figure 5). Control sample pawl nih benzoate metabolism (ko:K22270), nitrogen metabolism (ko:K00368), le glycolysis/gluconeogenesis (ko:K00131) he aa pehtlaimi genes tam deuh an ngei ( Figure 5 ).
Fig. 5. PPA le control mice hna i an paw chung microbiome ah KOs a tamnak aa dannak. Meitlang hmanthlak nih riantuannak phu (KOs) tamnak aa dannak a langhter. A senmi dot nih KOs a langhter i an tamnak cu sample phun karlak ah aa dang tuk lo (p-value > 0.05). A rong aa dawhmi dot nih a tamnak ah a biapi mi thleidannak a langhter (p-value ≤ 0.05). Sample phun karlak ah a tam bik aa dannak a ngeimi KO 20 cu control le PPA sample he aa tlakmi sen le sen rang in langhter an si. A senmi le a senmi dot nih KOs cu control le PPA sample ah a tlawmbik 2.7-fold in a tam deuh ti kha a langhter. A senmi dot nih KOs cu a tam ning aa dang tukmi a langhter, -1 le 1 karlak ah a tlangpi in CLR aa dannak he. P man cu Mann-Whitney U hneksaknak hmangin tuak a si i Benjamini-Hochberg phunglam hmangin tahchunhnak tampi caah remh a si. NaN nih KO cu KEGG chung i lam pakhatkhat ah aa tel lo ti a langhter. Bold mean CLR thleidannak man nih tamnak ah a biapi mi thleidannak a langhter. Cazin ah aa telmi KO pawl an i telnak lam kong he pehtlai in a tawinak in theih na duh ahcun, Supplementary Table 3 zoh.
Annotated genes hna lakah, 1601 genes nih sample phun karlak ah aa dang tukmi an ngei (p ≤ 0.05), gene pakhat cio cu a tlawmbik 2.7-fold in an tam deuh. Hi genes hna chungin, genes 4 cu control sample ah an tam deuh i genes 1597 cu PPA sample ah an tam deuh. PPA nih rungrul thah khawhnak a ngeih caah, sample phun karlak ah PPA thlennak le chuahnak genes a tamnak kha kan zoh. PPA thlennak he aa pehtlaimi gene 1332 chungin, gene 27 cu control sample ah a tam deuh ngaingai i PPA sample ah gene 12 cu a tam deuh. PPA chuahnak he aa pehtlaimi gene 223 lakah, gene 1 cu PPA sample ah a tam deuh ngaingai. Figure 6A nih PPA thlennak ah aa telmi genes a tam deuhmi kha a langhter chin, control sample ah a tam deuhmi le a nganmi thlennak ngan he, cu lioah Figure 6B nih PPA sample ah hmuhmi a tam deuhmi genes pakhat cio kha a langhter.
Fig. 6. Mouse gut microbiome chung i PPA he aa pehtlaimi genes a tamnak aa dangmi. Meitlang hmanthlak nih PPA thlennak (A) le PPA chuahnak (B) he aa pehtlaimi genes tamnak aa dannak kha a langhter. A senmi dot nih sample phun karlak ah a tamnak aa dang lomi genes a langhter (p-value > 0.05). A rong aa dawhmi dot nih a tamnak ah a biapi mi thleidannak a langhter (p-value ≤ 0.05). A tam bik aa dannak a ngeimi gene 20 cu a sen le a sen (control le PPA sample) in langhter an si. A senmi le a senmi dot tamnak cu control le PPA sample ah control sample nakin a tlawmbik let 2.7 in a tam deuh. A senmi dot nih a tlawmtam aa dang tukmi genes a aiawh, CLR aa dannak cu -1 le 1 karlak a si. P man cu Mann-Whitney U hneksaknak hmangin tuak a si i Benjamini-Hochberg phunglam hmangin tahchunhnak tampi caah remh a si. Genes cu a herh lomi gene cazin chung i aiawhtu genes he aa tlak. Gene min hna cu KO gene a langhtermi KEGG hmelchunhnak in an um. Bold mean CLR aa dannak nih aa dang tukmi tamnak a langhter. Dash (-) nih KEGG database ah gene caah hmelchunhnak a um lo ti a langhter.
PPA thlennak le/asiloah chuahnak he aa pehtlaimi genes a ngeimi taxa cu contigs i an taxonomic sining le gene i an contig ID kha tahchunh in hngalh khawh an si. Genus level ah, phun 130 cu PPA thlennak he aa pehtlaimi genes an ngei i phun 61 cu PPA chuahternak he aa pehtlaimi genes an ngei tiah hmuh an si (Supplementary Table 4). Sihmanhsehlaw, phun pakhat hmanh nih a tamnak ah a biapi mi thleidannak an langhter lo (p > 0.05).
Phunphun tlukruannak ah, PPA thlennak he aa pehtlaimi genes a ngeimi bacteria phun 144 le PPA chuahternak he aa pehtlaimi genes a ngeimi bacteria phun 68 an hmuh hna (Supplementary Table 5). PPA thlennak hna lakah, bacteria pariat nih sample phun karlak ah a karh ning a langhter, cun an dihlak in thlennak a langhter (Supplementary Table 6). A tamnak ah aa dangmi hmuhmi PPA metabolizers vialte cu PPA sample ah an tam deuh. Species-level thleidannak nih Bacteroides le Ruminococcus phun cheukhat, cun Duncania dubois, Myxobacterium enterica, Monococcus pectinolyticus, le Alcaligenes polymorpha hna telh in sample phun karlak ah aa dang tukmi phun aiawhtu a langhter. PPA a chuahtertu bacteria hna lakah, bacteria pali nih sample phun karlak ah a tamnak ah a biapi mi thleidannak an langhter. A tam ning aa dangmi phun hna cu Bacteroides novorossi, Duncania dubois, Myxobacterium enteritidis, le Ruminococcus bovis hna an si.
Hi hlathlainak ah, PPA nih vok pawl an pawpi chung thilnung hna cungah a chuahpimi thil kha kan zoh. PPA nih hin bacteria ah lehnak phunphun a chuahter khawh, zeicahtiah phun cheukhat nih an chuahmi a si, phun dang nih rawl ah an hman, asiloah antimicrobial tthatnak a ngei. Cucaah, rawl chapnak in pawpi pawngkam ah a chapmi nih tuar khawhnak, tuar khawhnak le rawlchuannak hrampi ah hman khawhnak cungah aa hngatmi thlennak a chuahpi kho. A tthawngmi bacteria phun pawl cu hloh khawh an si i PPA a doh khawh deuhmi asiloah rawl caah hman khawhmi nih an thlen khawh hna, cu nih cun pawpi chung thilnung hme tete hna i an i fonhnak ah thlennak a chuahter. Kan hmuhmi nih hin microbial fonhnak ah a biapi mi thleidannak a langhter nain microbial phunphun ah zeihmanh thlennak a chuahpi lo. A ngan bikmi thlennak cu saram phun level ah hmuh a si, PPA le control sample karlak ah 70 leng taxa cu a tamnak ah aa dang ngaingai (Supplementary Table 2). PPA-a tongmi sample pawl an i fonhnak zohfelnak nih a langhter lomi sample pawl he tahchunh tikah microbial phun hna i an i dannak a tam deuh ti a langhter, cu nih cun PPA nih bacteria ṭhanchonak sining a ṭhanchoter khawh i PPA-a tammi pawngkam ah a nung khomi bacteria milu kha a tlawmter khawh ti kha a langhter. Cucaah, PPA nih hin gut microbiota phunphun ah a karhmi buainak a chuahter nakin thlennak kha a thim tein a chuahter khawh.
PPA tibantuk rawl kilvennak nih a hlan ah a phunphun in a ummi hna kha hnahnawhnak pe loin pawpi chung i a ummi thilnung hna an tamnak kha a thlen khawh tiah an rak langhter cang (Nagpal et al., 2021). Hika ah, Bacteroidetes phun (a hlan ah Bacteroidetes tiah theihmi) chung i Bacteroidetes phun hna karlak ah a lang ngaimi thleidannak kan hmuh, cu hna cu PPA-a tongmi vok ah a ttha ngaimi an si. Bacteroides phun an karh ahcun zunthlum rawhnak a karh, cu nih cun zawtnak hrik a karhter khawh i zawtnak a chuahter khawh (Cornick et al., 2015; Desai et al., 2016; Penzol et al., 2019). Hlathlainak pakhat nih Bacteroides fragilis in thlopmi hrinmi pa vok hna nih autism spectrum zawtnak (ASD) bantuk in zatlang nun ziaza an langhter ti a hmuh (Carmel et al., 2023), cun hlathlainak dang nih Bacteroides phun nih pumsa ralvennak cawlcanghnak a thlen khawh i mah le mah i dohnak lung zawtnak a chuahter khawh ti a langhter (Carmel et al., Carmel et al., 2023). 2019). Ruminococcus, Prevotella, le Parabacteroides phun chung i aa telmi phun hna zong PPA a tongmi vok hna ah an karh ngaingai (Coretti et al., 2018). Ruminococcus phun cheukhat cu proinflammatory cytokines chuahternak thawngin Crohn zawtnak bantuk zawtnak he pehtlaihnak an ngei (Henke et al., 2019), cu lio ah Prevotella phun Prevotella humani bantuk cu thikai le insulin sensitivity tibantuk metabolic zawtnak he pehtlaihnak an ngei (Pedersen et al., 2016; Li et al., 2017). A donghnak ah, Bacteroidetes (a hlan ah Firmicutes tiah theihmi) le Bacteroidetes zat cu PPA-a tongmi vok ah control vok nakin a niam deuh ti kan hmuh, zeicahtiah Bacteroidetes phun an tam deuh caah a si. Hi ratio hi a hlan ah pawpi sining a langhtertu a biapi mi a si tiah langhter a si cang, cun hi ratio ah buainak a chuah tikah zawtnak phunphun he pehtlaihnak a ngei (Turpin et al., 2016; Takezawa et al., 2021; An et al., 2023), cu chung ah cun pawpi zawtnak zong aa tel (Stojanov et al., 2020). A dihlak in, phylum Bacteroidetes phun pawl cu a sangmi rawl PPA nih a tlunh bikmi an lo. Mah cu PPA tuar khawhnak a sang deuh caah asiloah PPA kha thazaang chuahnak ah hman khawhnak ruangah a si kho, cucu a tlawmbik ah phunkhat, Hoylesella enocea (Hitch et al., 2022) caah a hmaan tiah langhter a si. A dang tein, nu nih PPA a tonmi nih nauinn ṭhanchonak kha a ṭhanchoter khawh i vok fa hna an pawpi kha Bacteroidetes colonization ah a tuar khawh deuhmi a siter khawh; sihmanhsehlaw, kan hlathlainak suaisam nih cu bantuk thlennak cu a onh lo.
Metagenomic content zohfelnak nih PPA thlennak le chuahnak he aa pehtlaimi genes tamnak ah a biapi mi thleidannak a langhter, PPA-a tongmi vok nih PPA chuahternak caah tuanvo a ngeimi genes tamnak a langhter, cu lioah PPA-a tong lomi vok nih PAA metabolism caah tuanvo a ngeimi genes tamnak a langhter (Figure 6). Hi hmuhmi nih hin PPA nih microbial composition cung ah a chuahpimi thil cu a hmannak lawng ruangah a si kho men lo ti a langhter, cu lo ahcun PPA metabolism he aa pehtlaimi genes tamnak nih PPA-a tongmi vok hna i an pawpi microbiome ah a tam deuhmi a langhter awk a si. A fianternak pakhat cu PPA nih bacteria nih rawl ah a hmanmi nakin a bik in a antimicrobial tthatnak thawngin bacteria tamnak a thlen. Hlan lio hlathlainak nih PPA nih Salmonella Typhimurium tthanchonak kha a tlawmtam ning in a kham ti a langhter (Jacobson et al., 2018). PPA a tam deuhmi he i tonnak nih a rungrul thah khawhnak a doh khomi bacteria kha a thim khawh i a thlen khawh asiloah a chuah khawh hrimhrim lo. Tahchunhnak ah, Parabacteroides phun zeimawzat nih PPA sample ah a tam deuhmi an langhter, asinain PPA thlennak le chuahnak he aa pehtlaimi gene pakhat hmanh an hmu lo (Supplementary Tables 2, 4, le 5). Cun, PPA chuahmi cu rawlchuannak in a chuakmi thil pakhat a si i bacteria phunphun lakah a karh ngaingai (Gonzalez-Garcia et al., 2017). Bacteria phunphun a tam deuhmi cu control sample ah PPA metabolism he aa pehtlaimi genes a tam deuhmi a ruang a si kho (Averina et al., 2020). Cun, 1332 genes chungin 27 (2.14%) lawng hi PPA metabolism he aa pehtlaimi genes an si lai tiah an rak chimchung. PPA thlennak he aa pehtlaimi gene tampi cu thlennak lam dang zongah an i tel ve. Mah nih hin PPA thlennak ah aa telmi genes tamnak cu control sample ah a tam deuh ti kha a langhter chin; hi genes pawl nih hin PPA hmannak asiloah a chuakmi thil pakhat bantuk in sernak a chuahter lomi lam ah rian an ttuan kho. Hi kongah, PPA chuahternak he aa pehtlaimi gene pakhat lawng nih sample phun karlak ah a tamnak ah a biapi mi thleidannak a langhter. PPA thlennak he aa pehtlaimi genes he aa dang in, PPA chuahternak caah hmelchunhnak genes cu PPA chuahternak caah bacteria lam ah direct in an i tel caah thim an si. PPA-a tongmi vok hna ah, phun vialte nih PPA chuah khawhnak le an tamnak a karh ngaingai ti an hmuh. Mah nih hin PPAs nih PPA chuahtu an thim hna lai timi chimchungnak kha a dirpi i cucaah PPA chuah khawhnak a karh lai tiah an chimchung. Sihmanhsehlaw, gene tamnak cu gene langhternak he aa pehtlai hrimhrim lo; cucaah, PPA thlennak he aa pehtlaimi genes tamnak cu control sample ah a tam deuh ko nain, langhternak rate cu aa dang kho (Shi et al., 2014). PPA-chuahtertu genes a karh ning le PPA chuahternak karlak pehtlaihnak fehternak caah, PPA chuahternak ah aa telmi genes langhternak kong hlathlainak tuah a herh.
PPA le control metagenomes hna i riantuannak fianternak nih aa dannak cheukhat a langhter. PCA nih gene chung thilri hlathlainak nih PPA le control sample karlak ah aa dangmi phu pawl a langhter (Figure 5). Sample chung i fonhnak nih control gene a ummi cu a phunphun a si ti a langhter, PPA sample pawl cu hmunkhat ah an i fonh. Gene chunglei in fonh cu phun fonh in fonh he tahchunh khawh a si. Cucaah, lamthluan tamnak ah aa dannak cu an chung i a ummi phun le phunkhat tamnak ah thlennak he aa tlak. PPA sample ah, a tam deuhmi lam pahnih cu aminosugar/nucleotide sugar thlennak (ko:K21279) le lipid thlennak lam tampi (ko:K00647, ko:K03801; Supplementary Table 3) he aa pehtlaimi an si. ko:K21279 he aa pehtlaimi genes cu PPA sample ah a tam deuhmi phun pakhat a simi Bacteroides phun he aa pehtlaimi an si ti theih a si. Hi enzyme nih hin capsular polysaccharides (Wang et al., 2008) a langhternak thawngin pumsa ralvennak kha a hrial khawh. Mah nih hin PPA-a tongmi vok ah hmuhmi Bacteroidetes a karhnak a ruang a si kho men. Mah nih hin PPA microbiome ah hmuhmi a karhmi thau acid sernak kha a bawmh. Bacteria nih FASIIko:K00647 (fabB) lam kha thau acid chuah awkah an hman, cu nih cun a ngeitu metabolic lam kha a thlen khawh (Yao le Rock, 2015; Johnson et al., 2020), cun lipid metabolism thlennak nih thluak tthanchonak ah hmunhma a lak khawh (Yu et al., 2020). PPA sample ah a karhmi a langhtermi lam dang pakhat cu steroid hormone biosynthesis (ko:K12343) a si. Gut microbiota nih hormone dat a thlen khawhnak le hormone nih a thlen khawhnak karlak ah aa ralchanhmi pehtlaihnak a um ti langhternak a karh chin lengmang, cucaah steroid dat a sangmi nih ngandamnak lei ah a ttha lomi thil a chuahpi khawh (Tetel et al., 2018).
Hi hlathlainak hi rikhiahnak le ruahnak cheuhnak a um lo. A biapi mi thleidannak cu saram hna an pumsa lei zohfelnak kan tuah lo. Cucaah, microbiome thlennak cu zawtnak he aa pehtlaimi a si maw ti cu direct in biachahnak tuah khawh a si lo. Ruah awk dang pakhat cu hi hlathlainak ah aa telmi vok pawl cu an nu he aa khatmi rawl an pek hna. Hmailei hlathlainak nih PPA a tammi rawl in PPA a ngei lomi rawl ah thlennak nih microbiome cung ah a tthatnak a tthanchoter maw ti kha a hngalh khawh. Kan hlathlainak i rikhiahnak pakhat cu, a dang tampi bantuk in, sample tlawmte lawng a si. A hmaanmi biachahnak tuah khawh a si ko nain, a ngan deuhmi sample nih a phichuak hna kha hlathlainak tuah tikah zatlangh khawhnak thazaang a pek deuh lai. Cun, pawpi chung thilnung hme tete thlennak le zeibantuk zawtnak hmanh karlak pehtlaihnak kongah biachahnak tuah tikah kan i ralring (Yap et al., 2021). Kum, nu le pa sinak, le rawl eidin telh in a buaitertu thil hna nih thilnung hme tete an i fonh ning kha tampi in a thlen khawh. Hi thil hna nih hin pawpi chung thilnung hme tete le a harmi zawtnak hna pehtlaihnak kong he pehtlai in cauk chung ah hmuhmi aa kalhmi thil hna kha a fianter khawh (Johnson et al., 2019; Lagod le Naser, 2023). Tahchunhnak ah, Bacteroidetes phun chungtel hna cu ASD a ngeimi saram le minung ah an karh asiloah an zor ti a si (Angelis et al., 2013; Kushak et al., 2017). Cu bantuk cun, zunthlum zawtnak a ngeimi mizaw hna i an pawpi sining kong hlathlainak nih taxa pakhat ah a karh le a zor an hmuh (Walters et al., 2014; Forbes et al., 2018; Upadhyay et al., 2023). Nu le pa thleidannak nih a chuahpimi thil kha tlawmter awkah, nu le pa tlukruannak aiawhnak kha kan i zuam i cuticun aa dannak cu rawl eidin nih a chuahter deuh lai. Riantuannak fianternak ah harnak pakhat cu a herh lomi gene zulhphung pawl hloh kha a si. Kan gene fonhnak phunglam nih 95% zulhin aa khatmi le 85% saunak aa lawhnak a herh, cun a hmaan lomi fonhnak hloh khawhnak ding caah 90% tlukruannak khuh a herh. Sihmanhsehlaw, thil sining cheukhat ahcun, aa khatmi fianternak (tahchunhnak ah, MUT) he COGs kan hmuh (Fig. 6). Hi orthologs pawl hi aa dang maw, phun pakhatkhat he aa pehtlaimi an si maw, asiloah hi cu gene fonhnak lamthluan ah rikhiahnak a si maw ti hngalh khawhnak ding caah hlathlainak tuah chap a hau. Riantuannak fianternak i rikhiahnak dang pakhat cu thleidannak palh a si khomi a si; bacteria gene mmdA cu propionate sernak ah aa telmi theihmi enzyme a si, asinain KEGG nih propionate thlennak lam he a pehtlaihter lo. Cu he aa dang in, scpB le mmcD orthologs cu an i pehtlai. Knockouts a um lomi genes tampi nih genes tamnak tuaktan tikah PPA he aa pehtlaimi genes hngalh khawh lonak a chuahter khawh. Hmailei hlathlainak nih metatranscriptome hlathlainak in ṭhatnak a hmuh lai, mah nih cun pawpi chung thilnung hme tete hna i rianṭuannak sining hna kha thukpi in hngalhthiamnak a pek khawh hna lai i gene langhternak kha a tanglei thil sining hna he a pehtlaihter khawh hna lai. A bik in thluak tthanchonak lei zawtnak asiloah zunthlum zawtnak he aa pehtlaimi hlathlainak hna caah, saram hna i an pumsa le an ziaza zohfelnak hna cu hi zawtnak hna he microbiome fonhmi thlennak hna pehtlaih khawhnak ding caah a herh. Pawchung microbiome kha rungrul a ngei lomi vok ah thlennak kong hlathlainak dang zong nih microbiome cu zawtnak a chuahtertu asiloah a sining a si le si lo hngalh khawhnak caah a ttha lai.
A tawinak in, rawl eidin PPA nih pawpi chung thilnung hme tete a thlennak ah thil pakhat in rian a ttuan ti kha kan langhter. PPA cu FDA nih a cohlanmi kilvennak sii a si i rawl phunphun ah tampi hmuh khawh a si i, caan saupi hman ahcun, a ttha mi pawpi chung thilnung hna a hrawh khawh. Bacteria zeimawzat an tamnak ah thlennak kan hmuh, cu nih cun PPA nih pawpi microbiota dirhmun a thlen khawh ti a langhter. Microbiota thlennak nih metabolic lam cheukhat thlennak a chuahter khawh, cu nih cun inntek ngandamnak he aa pehtlaimi pumsa lei thlennak a chuahter khawh. Rawl eidin PPA nih microbial composition cung ah a chuahpimi thil nih dysbiosis asiloah zawtnak dang a chuahter khawh maw ti hngalh khawhnak ding caah hlathlainak tuah chap a hau. Hi hlathlainak nih hin hmailei ah PPA nih pawpi chung thilri cung ah a chuahpimi thil nih minung ngandamnak a hnorsuan khawh ning kong hlathlainak caah hrampi a chiah.
Hi hlathlainak ah langhtermi dataset pawl cu online chiahnak hmun ah hmuh khawh an si. Chiahnak hmun min le luh khawhnak nambar cu: https://www.ncbi.nlm.nih.gov/, PRJNA1092431.
Hi saram hlathlainak cu University of Central Florida Institutional Animal Care and Use Committee (UCF-IACUC) nih an cohlan (Saram hmannak nawl nambar: PROTO202000002). Hi hlathlainak nih hin khuachung upadi, phunglam le bupi nih an herhmi he aa tlak.
NG: Ruahnak cheuhnak, Data zohkhenhnak, Phungning tein hlathlainak, Hlathlainak, Lamthluan, Software, Hmuh khawhnak, Catialnak (a hramthawk suaimi), Catialnak (zohfelnak le remhnak). LA: Ruahnak cheuhnak, Data zohkhenhnak, Lamthluan, Hmanmi thilri, Catialnak (zohfelnak le remhnak). SH: Phungning tein hlathlainak, Software, Catialnak (zohfelnak le remhnak). SA: Hlathlainak, Catialnak (zohfelnak le remhnak). Biaceihtu Bawi: Hlathlainak, Catialnak (zohfelnak le remhnak). SN: Ruahnak cheuhnak, Project uknak, Thilri, Zohkhenhnak, Catialnak (zohfelnak le remhnak). TA: Ruahnak cheuhnak, Project uknak, Zohkhenhnak, Catialnak (zohfelnak & remhnak).
Catialtu hna nih hi capar hlathlainak, catialtu sinak le/asiloah chuahnak caah tangka bawmhnak an hmu lo tiah an thanh.
Catialtu hna nih hlathlainak cu chawlehthalnak le phaisa lei pehtlaihnak a um lonak hmun ah tuahmi a si tiah an thanh. hman khawh a si lo.
Hi capar ah langhtermi ruahnak vialte cu catialtu hna ruahnak lawng an si i an bu, chuahtu, editor, asiloah zohfeltu hna ruahnak kha a langhter hrimhrim lo. Hi capar ah zohfelmi thilri paohpaoh, asiloah an sertu hna nih an chimmi paohpaoh cu chuahtu nih a fehtermi asiloah a cohlanmi an si lo.
Hi capar caah bawmtu thilri cu online ah hmuh khawh a si: https://www.frontiersin.org/articles/10.3389/frmbi.2024.1451735/full#supplementary-material
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