Say propionic acid (PPA), sakey ya antifungal ya ahente tan kaslakan ya kaaruman ed panangan, et napatnagan ya pansengegan na abnormal ya neurodevelopment na saray ayep ya kaiba so gastrointestinal dysfunction, ya nayarin sengegan na gut dysbiosis. Walay nisuheri ya siglaotan na pakawalaan na PPA ed panangan tan say gut microbiota dysbiosis, balet ag ni direktan naimbestigaan. Dia, inimbestigaan mi ray pananguman ed komposisyon na gut microbiota ya misiglaotan ed PPA ya nayarin pansengegan na dysbiosis. Saray gut microbiomes na saray ayep ya pinakan na ag-atambal a naakan (n=9) tan naakan ya walay PPA to (n=13) et in-sequence ya inusar so long-range metagenomic sequencing pian nanengneng so pandumaan na komposisyon na mikrobyo tan saray dalan na metabolismo na bakterya. Say PPA ed kakanen et walay koneksion to ed idarakel na karakel na makabkabaliksan iran taxa, kaiba lay pigaran klase na Bacteroides, Prevotella, tan Ruminococcus, ya saray membro da et datin nalalanor ed produksyon na PPA. Saray microbiomes na PPA-exposed mice et walaan met na mas dakel a dalan ya misiglaotan ed lipid metabolism tan steroid hormone biosynthesis. Ipapanengneng na resulta mi ya say PPA et nayarin manguman ed gut microbiota tan saray metabolic pathways ya kaiba to. Ipapabitar na sarayan naobserbaan ya pananguman ya saray preserbatibo ya niklasipika ya ligtas ya kanen et nayarin mangimpluensya ed komposisyon na gut microbiota tan, diad saginonor, say bunigas na too. Diad saratan, say P, G o S so napili depende ed lebel na klasipikasyon ya aanalisaen. Pian nabawasan so epekto na saray palson positibon klasipikasyon, inadapta so sankaabebaan ya relative abundance threshold ya 1e-4 (1/10,000 reads). Antis na statistical analysis, saray relatibon karakel ya inreport nen Bracken (fraction_total_reads) et inuman ya inusar so centered log-ratio (CLR) transformation (Aitchison, 1982). Say CLR ya paraan so pinili parad data transformation lapud ag-itan manguuman ed sukat tan magenap parad saray aliwan sparse ya dataset (Gloor et al., 2017). Say CLR ya pananguman et manguusar na natural ya logarithm. Say bilang na datos ya inreport nen Bracken et in-normalize ya inusar so relative log expression (RLE) (Anders tan Huber, 2010). Saray pigura et ginawa ya inusar so kombinasyon na matplotlib v. 3.7.1, seaborn v. 3.7.2 tan sequential logarithms (Gloor et al., 2017). 0.12.2 tan stantanotations v. 0.5.0 (Hunter, 2007; Waskom, 2021; Charlier et al., 2022). Say ratio na Bacillus/Bacteroidetes et kinalkula parad kada sample ya inusar so normalisadon bilang na bakterya. Saray balor ya nireport ed saray table et nabilog ed 4 a decimal places. Say Simpson diversity index et kinalkula ya inusar so alpha_diversity.py script ya inter ed KrakenTools v. 1.2 package (Lu et al., 2022). Say Bracken report et niiter ed script tan say Simpson index ya “Si” et niiter parad -an parameter. Saray makabkabaliksan a pandumaan ed karakel so nidepinsa bilang average a pandumaan na CLR ≥ 1 odino ≤ -1. Say average ya pandumaan na CLR ya ±1 et mangipapanengneng na 2.7-fold ya indaak na karakel na sakey a klase na sample. Say tanda (+/-) et mangipapanengneng no kasin mas dakel so taxon ed PPA sample tan control sample, unong ed inkauksoy da. Say kabaliksan to et nadetermina panamegley na Mann-Whitney U test (Virtanen et al., 2020). Say Statsmodels v. 0.14 (Benjamini tan Hochberg, 1995; Seabold tan Perktold, 2010) so inusar, tan say Benjamini-Hochberg a pamaakaran so inyaplika pian ipetek so dakel a panagsukimat. Say inuman ya p-value ≤ 0.05 so inusar bilang threshold parad pakadetermina na statistical significance.
Say microbiome na too et mabetbet ya tatawagen ya “say unor ya organo na laman” tan walay importantin betang to ed bunigas na too (Baquero tan Nombela, 2012). Diad partikular, say gut microbiome et kabkabat lapud impluensya to ed interon sistema tan papel to ed dakel ya importantin kimey. Saray commensal bacteria et dakel ed bituka, ya mangookupa na nanduruman pasen ed ekolohiya, manguusar na sustansya, tan mikokompetensya ed saray posiblin pathogen (Jandhyala et al., 2015). Saray nanduruman kabiangan na bakterya ed gut microbiota et makapawala na saray importantin sustansya a singa saray bitamina tan mamaaligwas ed panagtunaw (Rowland et al., 2018). Saray metabolite na bakterya et napatnagan met ya mangiimpluensya ed itatakken na tissue tan mamaaligwas ed metabolic tan immune pathways (Heijtz et al., 2011; Yu et al., 2022). Say komposisyon na microbiome ed bituka na too et nanduruma tan mandedepende ed genetic tan environmental factors a singa say panangan, sekso, tambal, tan kipapasen na bunigas (Kumbhare et al., 2019).
Say panangan na ina et importantin kabiangan na itatakken na ugaw tan say kapangiyanak tan sakey a posiblin lapuan na saray compound ya nayarin mangimpluensya ed itatakken to (Bazer et al., 2004; Innis, 2014). Sakey ed saratan ya compound ya makapainteres et say propionic acid (PPA), sakey ya short-chain fatty acid by-product ya naala ed bacterial fermentation tan sakey ya food additive (den Besten et al., 2013). Say PPA et walaan na antibacterial tan antifungal ya kablian kanian uusaren bilang preserbatibo na naakan tan diad industriyal iran aplikasyon pian nasebel so amag tan idakel na bakterya (Wemmenhove et al., 2016). Say PPA et walaan na nanduruman epekto ed nanduruman tisyu. Diad altey, say PPA et walay anti-inflammatory ya epekto to diad pangapekta tod cytokine expression ed macrophages (Kawasoe et al., 2022). Sayan regulatory effect et naobserbaan met ed arum ya immune cells, ya pansengegan na downregulation na inflammation (Haase et al., 2021). Balet, say kabaliktaran ya epekto so naobserbaan ed utek. Impanengneng na saray akaunan impanaral ya say pakawalaan na PPA et mamawala na singa autism ya ugali ed saray ayep (El-Ansary et al., 2012). Impanengneng na arum ya impanaral ya say PPA et nayarin mamawala na gliosis tan mamakiwas ed saray pro-inflammatory pathways ed utek (Abdelli et al., 2019). Lapud say PPA et makapuy ya asido, nayarin onla ed intestinal epithelium ya onloob ed dala tan diad ontan et makalampas ed saray restrictive barriers kaiba lay blood-brain barrier tan say placenta (Stinson et al., 2019), ya mangipapabitar ed kaimportantian na PPA bilang sakey a regulatory metabolite ya gagawaen na bakterya. Anggaman say posiblin papel na PPA bilang sakey a risgo ed autism et kaplesan ya aaralen, say epekto to ed saray indibidual ya walay autism et nayarin onlaknab ni nen say pangigapo na neural differentiation.
Saray sintomas na gastrointestinal a singa say panag-ebak tan panag-ugip et kaslakan ed saray pasyente ya walay sakit ed neurodevelopmental (Cao et al., 2021). Impanengneng na saray akaunan impanaral ya say microbiome na saray pasyente ya walay autism spectrum disorders (ASD) et duma ed saray mabunigas ya indibidual, ya mangisusuheri na kiwawala na gut microbiota dysbiosis (Finegold et al., 2010). Ontan met, say microbiome characteristics na saray pasyente ya walay inflammatory bowel diseases, obesity, Alzheimer's disease, tan arum ni et midumaan met ed saray mabunigas ya indibidual (Turnbaugh et al., 2009; Vogt et al., 2017; Henke et al., 2019). Balet, anggad natan, anggapo ni so naimano ya siglaotan na gut microbiome tan saray sakit o sintomas na neurological (Yap et al., 2021), anggaman pigaran klase na bakterya so iisipen ya walay papel da ed pigara ed sarayan sakit. Singa bilang, say Akkermansia, Bacteroides, Clostridium, Lactobacillus, Desulfovibrio tan arum niran genera et mas dakel ed microbiota na saray pasyente ya walay autism (Tomova et al., 2015; Golubeva et al., 2017; Cristiano et al., 2018; Zurita et al., 2020). Say makatantanda, saray membro na pigara ed sarayan genera et kabkabat ya walaan na saray genes ya misiglaotan ed produksyon na PPA (Reichardt et al., 2014; Yun tan Lee, 2016; Zhang et al., 2019; Baur tan Dürre, 2023). Lapud antimicrobial properties na PPA, say pamarakep ed karakel to et nayarin makagunggona ed idakel na PPA-producing bacteria (Jacobson et al., 2018). Kanian, say PFA-rich ya kaliberliber et nayarin manresulta ed pananguman ed gut microbiota, kaiba lay gastrointestinal pathogens, ya nayarin posiblin sengegan na gastrointestinal sintomas.
Say manunan tepet ed panagsukimat ed microbiome et no kasin say pandumaan na komposisyon na mikrobyo et sengegan o sintomas na saray sakit. Say unonan kundang pian napalinew so komplikadon siglaotan na panangan, say gut microbiome, tan saray sakit ed utek et say pangusisa ed epekto na panangan ed komposisyon na mikrobyo. Diad sayan gagala, inusar mi so long-read metagenomic sequencing pian ikompara so gut microbiomes na saray anak na ayep ya pinakan na PPA-rich odino PPA-depleted ya kakanen. Saray anak et pinakan na parehon naakan a singa saray ina ra. Inisip mi ya say panangan ya dakel so PPA to et manresulta ed pananguman ed komposisyon na mikrobyo ed bituka tan saray dalan na mikrobyo, lalo la ed saray misiglaotan ed metabolismo na PPA tan/odino produksyon na PPA.
Sayan impanaral et angusar na FVB/N-Tg(GFAP-GFP)14Mes/J transgenic mice (Jackson Laboratories) ya man-overexpress na green fluorescent protein (GFP) diad silong na kontrol na glia-specific GFAP promoter ya ontutumbok ed saray totontonen na University of Central Florida Institutional Animal Care and Use Committee (UCIA) (UCIA) (UFUse Permit Number) PROTO202000002). Kayari na impan-wean, saray ayep so inyan ya indibidual ed saray kulungan ya walay 1-5 ya ayep na kada sekso ed kada kulungan. Saray ayep et pinakan na ad libitum na purified control diet (modified open-label standard diet, 16 kcal% fat) odino sodium propionate-supplemented diet (modified open-label standard diet, 16 kcal% fat, containing 5,000 ppm sodium propionate). Say karakel na sodium propionate ya inusar et mipara ed 5,000 mg PFA/kg ya kabuoan na belat na naakan. Saya so sankatageyan ya konsentrasyon na PPA ya inaprobaan ya usaren bilang preserbatibo na naakan. Pian makapanparaan ed sayan impanaral, saray atateng ya ayep so pinakan na parehon dieta ed loob na 4 simba antis na pan-mating tan intuloy da legan na panlukon na dam. Saray anak ya ayep [22 ya ayep, 9 ya kontrol (6 a lalaki, 3 a bibii) tan 13 a PPA (4 a lalaki, 9 a bibii)] so inekal insan intuloy so parehon panangan na saray dam ed loob na 5 bulan. Saray anak na ayep et insakripisyo nen 5 bulan so edad da tan saray karga na bituka ra et inala tan inmuna dan insimpen ed 1.5 ml ya microcentrifuge tubes ed -20°C insan inyalis ed -80°C ya freezer anggad naupot so DNA na host tan naekal iray microbial nucleic acids.
Say DNA na host et inekal unong ed sakey ya inuman ya protocol (Charalampous et al., 2019). Diad antikey, saray karga na dutak et inyalis ed 500 μl ya InhibitEX (Qiagen, Cat#/ID: 19593) tan insimpen ya akayelo. Iproseso so sankarakelan ya 1-2 ya fecal pellets kada panag-extract. Say karga na dutak et mekanikal ya in-homogenize ya inusar so plastik ya pestle ed loob na tubo pian magmaliw ya slurry. I-centrifuge iray sample ed 10,000 RCF ed loob na 5 min odino anggad napellet la ray sample, insan i-aspirate so supernatant tan i-resuspend so pellet ed 250 μl 1× PBS. Ilaktip so 250 μl 4.4% ya solusyon na saponin (TCI, numero na produkto S0019) ed sample bilang detergent pian nalukasan iray membrane na selula na eukaryotic. Saray sample et inlaok ya maong anggad nagmaliw ya makinis tan inkulongan ed temperatura na kuarto ed loob na 10 min. Insan, pian naderal iray eukaryotic cells, 350 μl ya danum ya anggapoy nuclease so inyarum ed sample, in-incubate ed loob na 30 s, insan 12 μl 5 M NaCl so inyarum. Saray sample et in-centrifuge ed 6000 RCF ed loob na 5 min. I-aspirate so supernatant tan i-resuspend so pellet ed 100 μl 1X PBS. Pian naekal so DNA na host, mangiyan na 100 μl HL-SAN buffer (12.8568 g NaCl, 4 ml 1M MgCl2, 36 ml ya danum ya anggapoy nuclease) tan 10 μl HL-SAN enzyme (ArticZymes P/N 70910-202). Saray sample et inlaok a maong panamegley na pipetting tan in-incubate ed 37 °C ed loob na 30 min ed 800 rpm ed EppendorfTM ThermoMixer C. Kayari na incubation, in-centrifuge ed 6000 RCF ed loob na 3 min tan inurasan na mamidua ed 800 μl tan 1000 μl X1 PBS. Diad kaunoran, i-resuspend so pellet ed 100 μl 1X PBS.
Say amin ya DNA na bakterya et inala ya inusar so New England Biolabs Monarch Genomic DNA Purification Kit (New England Biolabs, Ipswich, MA, Cat# T3010L). Say estandarten operating procedure ya inter na kit et medyo inuman. I-incubate tan mantinien so danum ya anggapoy nuclease ed 60°C antis ya operasyon parad pinal ya elution. Idagdag so 10 μl Proteinase K tan 3 μl RNase A ed kada sample. Insan idagdag so 100 μl na Cell Lysis Buffer tan ilaok ya maong. Saray sample et in-incubate ed EppendorfTM ThermoMixer C ed 56°C tan 1400 rpm ed loob na ag-onkulang ed 1 oras tan anggad 3 oras. Saray sample ya in-incubate et in-centrifuge ed 12,000 RCF ed loob na 3 minuto tan say supernatant manlapud kada sample et inyalis ed nikaduman 1.5 mL ya microcentrifuge tube ya walay 400 μL ya binding solution. Saray tubo et in-pulse vortex ed loob na 5-10 segundo ed 1 segundo ya interval. Iyalis so interon likidon karga na kada sample (manga 600-700 μL) ed sakey a filter cartridge ya inyan ed sakey a flow-through collection tube. Saray tubo et in-centrifuge ed 1,000 RCF ed loob na 3 minuto pian nawalaan na inmunan pan-binding so DNA insan in-centrifuge ed 12,000 RCF ed loob na 1 minuto pian naekal so nakekeraan a likido. Say sample column et inyalis ed balon collection tube insan inurasan na mamidua. Parad unonan panag-uras, mangiyan na 500 μL na wash buffer ed kada tubo. Ibaliw so tubo na 3-5 danay insan i-centrifuge ed 12,000 RCF ed loob na 1 minuto. Ibantak so likido ed tubo na panagkolekta tan ipawil so filter cartridge ed parehon tubo na panagkolekta. Parad komaduan panag-uras, mangiyan na 500 μL na wash buffer ed filter ya ag-ibaliw. Saray sample et in-centrifuge ed 12,000 RCF ed loob na 1 minuto. Iyalis so filter ed 1.5 mL LoBind® tube tan idagdag so 100 μL na pre-warmed ya anggapoy nuclease ya danum. Saray filter et in-incubate ed temperatura na kuarto ed loob na 1 minuto insan in-centrifuge ed 12,000 RCF ed loob na 1 minuto. Say eluted DNA et insimpen ed -80°C.
Say konsentrasyon na DNA et nasukat ya inusar so QubitTM 4.0 Fluorometer. Say DNA et imparaan ya inusar so QubitTM 1X dsDNA High Sensitivity Kit (Cat. No. Q33231) unong ed instruksion na manggagawa. Say inkaapag-apag na DNA fragment et sinukat ya inusar so AglientTM 4150 o 4200 TapeStation. Say DNA et imparaan ya inusar so AgilentTM Genomic DNA Reagents (Cat. No. 5067-5366) tan Genomic DNA ScreenTape (Cat. No. 5067-5365). Say panagparaan ed library et ginawa ya inusar so Oxford Nanopore TechnologiesTM (ONT) Rapid PCR Barcoding Kit (SQK-RPB004) unong ed instruksion na manggagawa. Say DNA et in-sequence ya inusar so ONT GridIONTM Mk1 sequencer ya walay Min106D flow cell (R 9.4.1). Saray sequencing settings et: high accuracy base calling, minimum q value ya 9, barcode setup, tan barcode trim. Saray sample et in-sequence ed loob na 72 oras, kayari na satan et insubmit so base call data parad kaaruman ya panagproseso tan panagsukimat.
Say panagproseso na bioinformatics et ginawa ya inusar iray akauna lan adeskribe a paraan (Greenman et al., 2024). Saray FASTQ file ya naala ed sequencing et inapag ed saray direktoryo parad kada sample. Antis na panagsukimat ed bioinformatics, say datos et inproseso ya inusar so onggendan a pipeline: unona, saray FASTQ file na saray sample et intipon ed saksakey a FASTQ file. Insan, saray nabasa ya mas antikey nen say 1000 bp et infilter ya inusar so Filtlong v. 0.2.1, ya say anguman labat ya parameter et say –min_length 1000 (Wick, 2024). Antis na kaaruman ya panagsalat, say kalidad na babasaen et nakontrol ya inusar so NanoPlot v. 1.41.3 ya walaan na sarayan parametro: –fastq –plots dot –N50 -o
Parad taxonomic classification, saray reads tan assembled contigs et inklasipikar ya inusar so Kraken2 v. 2.1.2 ( Wood et al., 2019 ). Manggawa na saray report tan output file parad saray babasaen tan saray pantitipon, unong ed inkauksoy da. Usaren so –use-names ya opsyon pian naanalisa iray nabasa tan natipon. Say –gzip-compressed tan –paired ya opsyon et nibaga parad saray nabasa ya segment. Say relatibon karakel na taxa ed metagenomes et kinalkula ya inusar so Bracken v. 2.8 ( Lu et al., 2017 ). Inmuna kamin nanggawa na kmer database ya walaan na 1000 bases ya inusar so bracken-build ya walaan na sarayan parametro: -d
Say gene annotation tan say relative abundance estimation et ginawa ya inusar so binago ya bersion na protocol ya indeskribe nen Maranga et al. (Maranga et al., 2023). Unona, saray contig ya mas antikey nen say 500 bp et inekal ed amin ya assembly ya inusar so SeqKit v. 2.5.1 (Shen et al., 2016). Saray pinilin asemblea et intipon ed sakey a pan-metagenome. Saray open reading frames (ORFs) et abitla ya inusar so Prodigal v. 1.0.1 (sakey a parallel version na Prodigal v. 2.6.3) ya walaan na sarayan parametro: -d
Saray genes so inmunan ingrupo unong ed Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog (KO) identifiers ya inter na eggNOG pian ikompara so karakel na gene pathway. Saray genes ya anggapoy knockouts odino saray genes ya walaan na dakel a knockouts et inekal antis ya analisaen. Say average ya karakel na kada KO ed kada sample et kinalkula tan ginawa so statistical analysis. Saray PPA metabolism genes et tinukoy bilang antokaman a gene ya inikdan na row ko00640 diad KEGG_Pathway column, ya mangipapanengneng na papel to ed propionate metabolism unong ed KEGG. Saray genes ya abitla ya misiglaotan ed produksyon na PPA et nilista ed Supplementary Table 1 (Reichardt et al., 2014; Yang et al., 2017). Ginawa iray permutation test pian naamtaan so metabolismo na PPA tan saray genes ed produksyon ya mas dakel ed kada klase na sample. Sanlibon permutation so ginawa parad kada gene ya inaral. Say p-value ya 0.05 so inusar bilang cutoff pian naamtaan so statistical significance. Saray functional annotations et niiter ed indibidual iran genes ed loob na sakey a cluster base ed saray annotations na saray representative genes ed loob na cluster. Saray taxa ya misiglaotan ed metabolismo na PPA tan/odino produksyon na PPA et nayarin nabidbir diad pangitunos ed saray contig ID ed saray output file na Kraken2 ed parehon contig ID ya nansiansia legan na functional annotation ya uusaren so eggNOG. Say significance testing et ginawa ya inusar so Mann-Whitney U test ya adeskribe la. Say panangipetek ed dakel a panagsukimat et ginawa ya inusar so Benjamini-Hochberg a paraan. Say p-value ya ≤ 0.05 so inusar bilang cutoff pian naamtaan so statistical significance.
Say pandurumaan na gut microbiome na saray ayep et inusisa ya inusar so Simpson diversity index. Anggapoy naobserbaan ya pandumaan na control tan PPA samples no nipaakar ed genus tan species diversity (p-value parad genus: 0.18, p-value parad species: 0.16) (Figure 1). Say komposisyon na mikrobyo et kinompara ya inusar so principal component analysis (PCA). Ipapanengneng na Figure 2 so inkagrupo na saray sample unong ed phyla da, ya mangipapanengneng ya wala ray pandumaan ed komposisyon na species na microbiomes ed baetan na PPA tan control samples. Sayan panagtipon et aliwan tanton napatnagan ed lebel na genus, ya mangisusuheri ya say PPA et mangaapekta ed pigaran bakterya (Supplementary Fig. 1).
Pigura 1. Alpha diversity na genera tan species ya komposisyon na mouse gut microbiome. Saray box plot ya mangipapanengneng na Simpson diversity indices na genera (A) tan species (B) ed PPA tan control samples. Say kabaliksan to et nadetermina ya inusar so Mann-Whitney U test, tan say multiple correction et ginawa ya inusar so Benjamini-Hochberg procedure. ns, say p-value et aliwan makabkabaliksan (p>0.05).
Pigura 2. Resulta na panagsukimat ed manunan kabiangan na komposisyon na mikrobyo ed bituka na mouse diad lebel na klase na ayep. Say plot na panagsukimat na manunan kabiangan et mangipapanengneng na inkaapag-apag na saray sample ed unonan duaran manunan kabiangan da. Saray kolor so mangipapanengneng na klase na sample: saray PPA-exposed ya ayep et kolor-ube tan saray kontrol ya ayep et duyaw. Saray manunan kabiangan 1 tan 2 et aka-plot ed x-axis tan y-axis, unong ed inkauksoy da, tan nibalikas ira bilang say impaliwawa dan variance ratio.
Diad pangusar na RLE transformed count data, walay anengneng ya signipikantin ibabawas na median Bacteroidetes/Bacilli ratio ed control tan PPA ya ayep (control: 9.66, PPA: 3.02; p-value = 0.0011). Sayan pandumaan et lapud mas dakel so Bacteroidetes ed PPA ya ayep no ikompara ed saray kontrol, anggaman aliwan makabkabaliksan so pandumaan (control mean CLR: 5.51, PPA mean CLR: 6.62; p-value = 0.054), bangbalet say karakel na Bacteroidetes et mipara (control mean CLR: 7.76, PPA-value: p-value: 0.066. = 0.18).
Say impanaral ed karakel na taxonomic members na gut microbiome et angipatnag ya 1 phylum tan 77 species so mandumaan a maong ed baetan na PPA tan control samples (Supplementary Table 2). Say karakel na 59 ya klase na ayayep ed PPA samples et mas atagey nen say control samples, bangbalet say karakel na 16 labat ya species ed control samples et mas atagey nen say PPA samples (Figure 3).
Pigura 3. Say pandumaan na karakel na taxa ed gut microbiome na PPA tan control mice. Saray volcano plot et mangipapanengneng na pandumaan na karakel na genera (A) odino species (B) ed baetan na PPA tan control samples. Saray abuek ya tuldok et mangipapanengneng ya anggapoy signipikantin pandumaan na karakel na taxa. Saray kolor ya tuldok et mangipapanengneng na signipikantin pandumaan ed karakel (p-value ≤ 0.05). Say sankatageyan a 20 taxa ya walaan na sankabalgan a pandumaan ed karakel na saray klase na sample et nipanengneng ed kolor ya ambalanga tan asul (control tan PPA samples), unong ed inkauksoy da. Saray duyaw tan lila ya tuldok et ag-onkulang ed 2.7 beses ya mas dakel ed saray sample odino PPA nen saray kontrol. Saray andeket a tuldok et mangirerepresenta na taxa ya walaan na signipikantin pandumaan na karakel, a walaan na mean CLR ya pandumaan ed baetan na -1 tan 1. Saray P values so kinalkula ya inusar so Mann-Whitney U test tan inkorihi parad dakel a panagsukimat ya inusar so Benjamini-Hochberg procedure. Say bold mean CLR ya pandumaan et mangipapanengneng na signipikantin pandumaan ed karakel.
Kayari min inaral so komposisyon na mikrobyo ed bituka, nanggawa kami na functional annotation na mikrobyo. Kayari na impangisalat ed saray abeban kalidad na genes, walay dagup ya 378,355 ya nikadkaduman genes ya abitla ed amin ya sample. Say inuman ya karakel na sarayan genes et inusar parad principal component analysis (PCA), tan say resulta et angipatnag na atagey a grado na panagtipon na saray klase na sample base ed saray functional profile da (Figure 4).
Figure 4. Resulta na PCA ya uusaren so functional profile na mouse gut microbiome. Ipapanengneng na PCA plot so inkaapag-apag na saray sample ed unonan duaran manunan kabiangan da. Saray kolor so mangipapanengneng na klase na sample: saray PPA-exposed ya ayep et kolor-ube tan saray kontrol ya ayep et duyaw. Saray manunan kabiangan 1 tan 2 et aka-plot ed x-axis tan y-axis, unong ed inkauksoy da, tan nibalikas ira bilang say nipaliwawa dan variance ratio.
Insan mi ineksamin so karakel na KEGG knockouts ed nanduruman klase na sample. Walay 3648 ya nikadkaduman knockouts ya abitla, ya 196 ed saratan so mas dakel ed saray control sample tan 106 so mas dakel ed saray PPA sample (Figure 5). Walay 145 ya genes ya anengneng ed saray control sample tan 61 ya genes ed saray PPA sample, ya walaan na signipikantin pandumaan na karakel. Saray dalan ya misiglaotan ed metabolismo na lipid tan aminosugar et mas mayaman ed saray sample na PPA (Supplementary Table 3). Saray dalan ya misiglaotan ed metabolismo na nitroheno tan saray sistema na sulfur relay et mas mayaman ed saray sample ya kontrol (Supplementary Table 3). Say karakel na genes ya misiglaotan ed aminosugar/nucleotide metabolism (ko:K21279) tan inositol phosphate metabolism (ko:K07291) et mas atagey ed saray sample na PPA (Figure 5). Saray sample ya kontrol et walaan na mas dakel a genes ya misiglaotan ed metabolismo na benzoate (ko:K22270), metabolismo na nitrogen (ko:K00368), tan glycolysis/gluconeogenesis (ko:K00131) ( Figure 5 ).
Fig. 5. Say pandumaan na karakel na KOs ed gut microbiome na PPA tan control mice. Say plot na bolkan so mangipapatnag na pandumaan na karakel na functional groups (KOs). Saray grey dots et mangipapanengneng na KOs ya say karakel da et aliwan signipikantin pandumaan ed baetan na saray klase na sample (p-value > 0.05). Saray kolor ya tuldok et mangipapanengneng na signipikantin pandumaan ed karakel (p-value ≤ 0.05). Say 20 KOs ya walaan na sankabalgan a pandumaan ed karakel na saray klase na sample et nipanengneng ed red tan light blue, ya mipara ed control tan PPA samples, unong ed inkauksoy da. Saray duyaw tan lila ya tuldok et mangipapanengneng na saray KO ya ag-onkulang ed 2.7-fold ya mas dakel ed control tan PPA samples, unong ed inkauksoy da. Saray andeket a tuldok so mangipapanengneng na saray KO ya walaan na signipikantin pandumaan na karakel, a walaan na average a pandumaan na CLR ed baetan na -1 tan 1. Saray P values so kinalkula ya inusar so Mann-Whitney U test tan in-adjust parad dakel a panangikompara ya inusar so Benjamini-Hochberg procedure. Ipapanengneng na NaN ya say KO et aliwan kabiangan na sakey a dalan ed KEGG. Say bold mean ya CLR ya pandumaan na balor et mangipapanengneng na signipikantin pandumaan ed karakel. Parad detalyadon impormasyon nipaakar ed saray dalan ya kawalaan na saray nilistan KO, nengnengen so Supplementary Table 3.
Diad saray annotated genes, 1601 ya genes so walaan na signipikantin pandumaan na karakel ed baetan na saray klase na sample (p ≤ 0.05), ya kada gene et ag-onkulang ed 2.7-fold ya mas dakel. Diad sarayan genes, 4 ya genes so mas dakel ed saray sample ya kontrol tan 1597 ya genes so mas dakel ed saray sample na PPA. Lapud say PPA et walaan na antimicrobial properties, ineksamin mi so karakel na PPA metabolism tan production genes ed baetan na saray klase na sample. Diad 1332 ya genes ya misiglaotan ed metabolismo na PPA, 27 ya genes so mas dakel ed saray sample ya kontrol tan 12 ya genes so mas dakel ed saray sample na PPA. Diad 223 ya genes ya misiglaotan ed produksyon na PPA, 1 gene so mas dakel ed saray sample na PPA. Ipapanengneng na Figure 6A so mas atagey ya karakel na genes ya nalalanor ed PPA metabolism, ya mas atagey so karakel to ed saray control sample tan baleg so epekto to, bangta say Figure 6B et mangipapabitar na indibidual iran genes ya mas atagey so naobserbaan ed saray sample na PPA.
Fig. 6. Say pandumaan na karakel na PPA-related genes ed mouse gut microbiome. Ipapanengneng na saray plot na bulkan so pandumaan na karakel na genes ya misiglaotan ed PPA metabolism (A) tan PPA production (B). Saray grey dots et mangipapanengneng na saray genes ya say karakel da et aliwan signipikantin pandumaan ed baetan na saray klase na sample (p-value > 0.05). Saray kolor ya tuldok et mangipapanengneng na signipikantin pandumaan ed karakel (p-value ≤ 0.05). Say 20 genes ya walaan na sankabalgan a pandumaan ed karakel et nipanengneng ed ambalanga tan light blue (control tan PPA samples), unong ed inkauksoy da. Say karakel na duyaw tan lila ya tuldok et ag-onkulang ed 2.7 beses ya mas dakel ed saray sample ya kontrol tan PPA nen saray sample ya kontrol. Saray andeket a tuldok so mangirerepresenta ed saray genes ya walaan na signipikantin pandumaan na karakel, a say average a pandumaan na CLR et walad baetan na -1 tan 1. Saray P values so kinalkula ya inusar so Mann-Whitney U test tan inkorihi parad dakel a panangikompara ya inusar so Benjamini-Hochberg procedure. Saray genes et mipara ed saray representative genes ed non-redundant gene catalog. Saray ngaran na gene et tugyopen na KEGG ya simbolo ya mangipapanengneng na KO gene. Say bold mean CLR ya pandumaan et mangipapanengneng na signipikantin pandumaan na karakel. Say dash (-) et mangipapanengneng ya anggapoy simbolo na gene ed KEGG database.
Saray taxa ya walaan na genes ya misiglaotan ed metabolismo tan/odino produksyon na PPA et nabirbir diad pangitunos ed taxonomic identity na contigs ed contig ID na gene. Diad genus level, 130 genera so aromog ya walaan na genes ya misiglaotan ed metabolismo na PPA tan 61 genera so aromog ya walaan na genes ya misiglaotan ed produksyon na PPA (Supplementary Table 4). Balet, anggapoy genera ya angipatnag na signipikantin pandumaan ed karakel (p > 0.05).
Diad lebel na species, 144 ya klase na bakterya so aromog ya walaan na genes ya mikonektaan ed metabolismo na PPA tan 68 ya klase na bakterya so aromog ya walaan na genes ya mikonektaan ed produksyon na PPA (Supplementary Table 5). Diad saray PPA metabolizers, waloran bakterya so angipatnag na signipikantin indaak na karakel ed baetan na saray klase na sample, tan amin da so angipatnag na signipikantin pananguman ed epekto (Supplementary Table 6). Amin ya naimano ya PPA metabolizers ya walay signipikantin pandumaan na karakel da et mas dakel ed saray sample na PPA. Say klasipikasyon ed lebel na klase so angipatnag na saray representante na genera ya ag tanton mandumaan ed baetan na saray klase na sample, kaiba lay pigaran klase na Bacteroides tan Ruminococcus, ontan met ed Duncania dubois, Myxobacterium enterica, Monococcus pectinolyticus, tan Alcaligenes polymorpha. Diad saray bakterya ya manggagawa na PPA, apatiran bakterya so angipatnag na signipikantin pandumaan ed karakel na saray klase na sample. Saray klase na ayayep ya walaan na baleg a pandumaan ed karakel et kaiba so Bacteroides novorossi, Duncania dubois, Myxobacterium enteritidis, tan Ruminococcus bovis.
Diad sayan impanaral, ineksamin mi so epekto na PPA exposure ed gut microbiota na saray ayep. Say PPA et makapangiter na nanduruman ebat ed bakterya lapud saya et gagawaen na pigaran klase na ayayep, uusaren na arum a klase na ayayep bilang naakan, odino walay epekto ton antimicrobial. Kanian, say pangiyarum to ed kaliberliber na bituka panamegley na dietary supplementation et nayarin walaan na nanduruman epekto depende ed tolerance, susceptibility, tan say abilidad ya usaren itan bilang lapuan na sustansya. Saray sensitibon klase na bakterya et nayarin naekal tan salatan na saramay mas makaresisti ed PPA odino sarag dan usaren itan bilang lapuan na naakan, ya pansengegan na pananguman ed komposisyon na gut microbiota. Say resulta mi et angipatnag na signipikantin pandumaan ed komposisyon na mikrobyo balet anggapoy epekto to ed inkalapagan a pandurumaan na mikrobyo. Say sankabalgan ya epekto et naobserbaan ed lebel na species, ya walay masulok ya 70 taxa ya signipikantin mandumaan ed karakel na PPA tan control samples (Supplementary Table 2). Say kaaruman ya impanaral ed komposisyon na saray sample ya inekspos ed PPA et angipatnag na mas baleg ya heterogeneity na saray klase na mikrobyo no ikompara ed saray sample ya ag-inekspos, ya mangisusuheri ya say PPA et nayarin mamaaligwas ed saray katangian na idakel na bakterya tan manlimitad populasyon na bakterya ya makapambilay ed saray kaliberliber ya mayaman ed PPA. Kanian, say PPA et nayarin manpili na pananguman imbes ya pansengegan na malaknab ya pakadisturbo na pandurumaan na gut microbiota.
Saray preserbatibo ed kakanen a singa say PPA et akauna lan nipanengneng ya manguuman ed karakel na saray kabiangan na gut microbiome ya ag-apektado so inkalapagan a pandurumaan (Nagpal et al., 2021). Dia, anengneng mi so sankabiglaan ya pandumaan na saray klase na Bacteroidetes ed loob na phylum ya Bacteroidetes (ya datin tatawagen ya Bacteroidetes), ya signipikantin inmayaman ed saray ayep ya akaeksperiensya na PPA. Say idakel na Bacteroides species et misiglaotan ed idakel na mucus degradation, ya nayarin mamaaligwas ed peligro na impeksion tan mamaaligwas na inflammation (Cornick et al., 2015; Desai et al., 2016; Penzol et al., 2019). Diad sakey ya impanaral, nalmoan ya saray neonatal ya lalakin ayep ya inikdan na Bacteroides fragilis et walaan na sosyal iran ugali ya mipara ed autism spectrum disorder (ASD) (Carmel et al., 2023), tan arum ya impanaral et angipatnag ya saray klase na Bacteroides et nayarin manguman ed immune activity tan pansengegan na autoimmune inflammatory cardiompathy (Carmel et al., Carmel et al., Carmel et al. 2019). Saray klase na klase na Ruminococcus, Prevotella, tan Parabacteroides et dinmaak met ed saray ayep ya akaekspos ed PPA (Coretti et al., 2018). Wala ray klase na Ruminococcus ya walay koneksion tod saray sakit a singa say Crohn's disease diad panamegley na produksyon na proinflammatory cytokines (Henke et al., 2019), bangbalet saray klase na Prevotella a singa say Prevotella humani et walay koneksion tod saray sakit ed metabolismo a singa say hypertension tan insulin sensitivity (Pedersen et al., 2016; Li et al., 2017). Diad kaunoran, aromog mi ya say ratio na Bacteroidetes (ya datin tatawagen ya Firmicutes) ed Bacteroidetes et mas abeba ed saray PPA-exposed ya ayep nen saray control ya ayep lapud mas atagey so dagup na Bacteroidetes species. Sayan ratio et datin nipanengneng ya importantin pakapatnagan na intestinal homeostasis, tan saray disturbo ed sayan ratio et nisiglaot ed nanduruman estado na sakit (Turpin et al., 2016; Takezawa et al., 2021; An et al., 2023), kaiba lay inflammatory bowel diseases (Stojanov et al., 2020). Diad inkalapagan, saray klase na phylum Bacteroidetes so ompatnag a mas apektado na atagey a PPA ed kakanen. Nayarin lapud mas atagey ya tolerance ed PPA odino say pakayarin mangusar na PPA bilang lapuan na enerhiya, ya apaneknekan ya tua parad anggan sakey a klase, say Hoylesella enocea (Hitch et al., 2022). Diad kasunian, say pakawalaan na PPA na ina et nayarin mamaaligwas ed itatakken na ugaw diad panggawa ed bituka na anak na mouse ya mas mainomay ya nakolonya na Bacteroidetes; balet, say desinyo na panagaral mi et ag-angiter na ontan ya panangusisa.
Say metagenomic content assessment et angipatnag na signipikantin pandumaan ed karakel na genes ya misiglaotan ed metabolismo tan produksyon na PPA, ya saray PPA-exposed mice et walaan na mas atagey a karakel na genes ya responsable ed produksyon na PPA, bangbalet saray non-PPA-exposed mice et walaan na mas atagey ya karakel na genes ya responsable ed PAA metabolism (Figure 6). Ipapanengneng na sarayan resulta ya say epekto na PPA ed komposisyon na mikrobyo et nayarin aliwan lapud panguusar to labat, ta no andi say karakel na genes ya misiglaotan ed metabolismo na PPA et nepeg ya mas dakel so napatnagan ed gut microbiome na saray ayep ya akaeksperiensya na PPA. Say sakey ya eksplinasyon et say PPA so mamapaarap ed karakel na bakterya diad panamegley na antimicrobial ya epekto to imbes ya diad panamegley na pangusar na bakterya bilang sustansya. Impanengneng na saray akaunan impanaral ya say PPA et mangaamper ed idakel na Salmonella Typhimurium diad paraan ya mandedepende ed dosis (Jacobson et al., 2018). Say pakawalaan na mas atagey a konsentrasyon na PPA et nayarin manpili na bakterya ya ag-onkukurang ed saray antimicrobial properties to tan nayarin agmet seguradon sarag ya i-metabolize o i-produce itan. Singa bilang, pigaran klase na Parabacteroides so angipatnag na mas atagey a karakel ed saray sample na PPA, balet anggapoy anengneng ya genes ya misiglaotan ed metabolismo odino produksyon na PPA (Supplementary Tables 2, 4, tan 5). Niarum ni, say produksyon na PPA bilang produkto na fermentation et malaknab ya nibunog ed nanduruman bakterya (Gonzalez-Garcia et al., 2017). Say mas atagey a pandurumaan na bakterya so nayarin rason ed mas atagey a karakel na genes ya misiglaotan ed metabolismo na PPA ed saray control sample (Averina et al., 2020). Sakey ni, 27 (2.14%) labat ed 1332 ya genes so naisip ya genes ya eksklusibon mikonektaan ed metabolismo na PPA. Dakel a genes ya misiglaotan ed metabolismo na PPA so nalalanor met ed arum niran dalan na metabolismo. Ipapanengneng na saya ya say karakel na genes ya nalalanor ed PPA metabolism et mas atagey ed saray control sample; sarayan genes et nayarin mankimey ed saray dalan ya agmanresulta ed pangusar odino pakaporma na PPA bilang byproduct. Diad sayan kaso, sakey labat a gene ya misiglaotan ed PPA generation so angipatnag na signipikantin pandumaan ed karakel na saray klase na sample. Diad pidumaan na saray genes ya misiglaotan ed metabolismo na PPA, saray marker genes parad produksyon na PPA so pinili lapud direktan nalalanor iratan ed dalan na bakterya parad produksyon na PPA. Diad saray ayep ya akaeksperiensya na PPA, amin ya klase na ayayep et aromog ya walaan na signipikantin indaak na karakel tan kapasidad dan mamawala na PPA. Susuportaan na saya so prediksion ya saray PPA so manpili na saray prodyuser na PPA kanian ipasakbay dan ondarakel so kapasidad na produksyon na PPA. Balet, say karakel na gene et agsigpot ya mikonektaan ed gene expression; kanian, anggaman say karakel na genes ya misiglaotan ed PPA metabolism et mas atagey ed saray control sample, say expression rate et nayarin duma (Shi et al., 2014). Pian nakumpirma so siglaotan na inkalaknab na saray genes ya manggagawa na PPA tan say produksyon na PPA, nakaukolan so panaral ed panangibalikas na saray genes ya nalalanor ed produksyon na PPA.
Say functional annotation na PPA tan control metagenomes et angipatnag na pigaran pandumaan. Say PCA analysis na gene content et angipatnag na nanduruman grupo ed baetan na PPA tan control samples (Figure 5). Say within-sample clustering et angipatnag ya mas nanduruma so control gene content, bangta saray PPA sample et nankakasakey. Say panagtipon base ed gene et mipara ed panagtipon base ed komposisyon na species. Kanian, say pandumaan na karakel na dalan et mipara ed pandurumaan na karakel na espisipikon klase tan strain ed loob da. Diad saray sample na PPA, duaran dalan ya mas atagey so karakel da et misiglaotan ed aminosugar/nucleotide sugar metabolism (ko:K21279) tan multiple lipid metabolism pathways (ko:K00647, ko:K03801; Supplementary Table 3). Saray genes ya misiglaotan ed ko:K21279 et kabkabat ya misiglaotan ed genus Bacteroides, sakey ed saray genera ya walaan na mas atagey a bilang na species ed saray sample na PPA. Sayan enzyme et makapaliis ed immune response diad pangibalikas na capsular polysaccharides (Wang et al., 2008). Nayarin saya so sengegan na indaak na Bacteroidetes ya naobserbaan ed saray ayep ya akaeksperiensya na PPA. Saya so mankokompleto ed indakel na fatty acid synthesis ya naobserbaan ed PPA microbiome. Uusaren na bakterya so FASIIko:K00647 (fabB) ya dalan pian makapawala na fatty acids, ya nayarin mangimpluensya ed saray dalan na metabolismo na host (Yao tan Rock, 2015; Johnson et al., 2020), tan saray pananguman ed metabolismo na lipid et nayarin walay papel to ed neurodevelopment (Yu et al., 2020). Say sananey nin dalan ya mangipapanengneng na indakel na saray sample na PPA et say steroid hormone biosynthesis (ko:K12343). Walay ondarakel ya ebidensya ya walay baliktaran a siglaotan na abilidad na gut microbiota ya mangimpluensya ed lebel na hormone tan say pakaimpluensya ed saray hormone, kanian say atagey ya lebel na steroid et nayarin walay epekto tod bunigas (Tetel et al., 2018).
Sayan panagaral et anggapoy limitasyon tan konsiderasyon to. Say importantin pandumaan et ag mi ginawa so physiological assessment na saray ayayep. Kanian, ag nayarin direktan ikonklusyon no kasin saray pananguman ed microbiome et walay koneksion tod antokaman a sakit. Say sananey nin ikonsidera et saray ayep ed sayan impanaral et pinakan na parehon kakanen na saray ina ra. Nayarin nadetermina na arapen iran panagaral no kasin say pangiyalis ed panangan ya dakel so PPA to ya anggad panangan ya anggapoy PPA et mamaaligwas ed epekto to ed microbiome. Sakey a limitasyon na impanaral mi, singa met ed arum, et say limitadon karakel na sample. Anggaman nayarin makagawa na balidon konklusyon, say mas baleg a sample size et mangiter na mas baleg ya statistical power no analisaen iray resulta. Maalwar kami met ed panggawa na konklusyon nipaakar ed siglaotan na pananguman ed gut microbiome tan antokaman a sakit (Yap et al., 2021). Saray makapawetwet a bengatla a singa say edad, sekso, tan panangan so nayarin makaimpluensya ed komposisyon na saray mikroorganismo. Sarayan bengatla so nayarin mangipaliwawa ed saray agpantatalosan ya naobserbaan ed literatura nipaakar ed siglaotan na gut microbiome ed saray komplikadon sakit (Johnson et al., 2019; Lagod tan Naser, 2023). Singa bilang, saray membro na genus Bacteroidetes et napatnagan ya ondarakel odino onbabawas ed ayayep tan totoon walay ASD (Angelis et al., 2013; Kushak et al., 2017). Ontan met, saray impanaral ed komposisyon na bituka na saray pasyente ya walay inflammatory bowel diseases et akaromog na parehon itatagey tan itatagey na parehon taxa (Walters et al., 2014; Forbes et al., 2018; Upadhyay et al., 2023). Pian nalimitaan so epekto na gender bias, sinali min seguroen so pantatalosan na saray sekso pian say pandumaan et posiblin nanlapud panangan. Sakey ya angat ed functional annotation et say pangekal ed saray redundant gene sequences. Say gene clustering method mi et mankaukolan na 95% ya sequence identity tan 85% ya length similarity, ontan met ed 90% ya alignment coverage pian naekal so false clustering. Balet, diad arum a kaso, anengneng mi ray COG ya parehoy annotation da (alimbawa, MUT) (Fig. 6). Kaukolan so kaaruman iran panagaral pian naamtaan no kasin sarayan orthologs et mandumaan, walay koneksion da ed espisipikon genera, odino no kasin saya so limitasyon na gene clustering approach. Say sananey nin limitasyon na functional annotation et say posiblin lingon panagklasipika; say bacterial gene mmdA et sakey a kabkabat ya enzyme ya walay koneksion tod propionate synthesis, balet say KEGG et agto ikokonekta ed propionate metabolic pathway. Diad pidumaan, say scpB tan mmcD orthologs et mankakasakey. Say karakel na genes ya anggapoy aturon knockouts et nayarin manresulta ed ag pakabidbiran na saray genes ya misiglaotan ed PPA no uusisaen so karakel na genes. Saray arapen ya panaral et nagunggonaan ed metatranscriptome analysis, ya makapangiter na mas aralem a pakatalos ed saray functional characteristics na gut microbiota tan mangikonekta ed gene expression ed saray posiblin epekto ed arapen. Parad saray panaral ya mangilalanor na espisipikon sakit ed neurodevelopmental odino inflammatory bowel diseases, nakaukolan so physiological tan behavioral assessments na ayayep pian nisiglaot iray pananguman ed komposisyon na microbiome ed sarayan sakit. Saray kaaruman ya panagaral ya pangiyalis na gut microbiome ed saray ayep ya anggapoy mikrobyo et makatulong met pian naamtaan no kasin say microbiome so sengegan odino pakabidbiran na sakit.
Diad sumaryo, impanengneng mi ya say PPA ed kakanen et onkikiwas bilang sakey a sengegan ed panguman na komposisyon na gut microbiota. Say PPA et sakey ya preserbatibo ya inaprobaan na FDA ya mabetbet ya naromog ed nanduruman kakanen ya no mabayag ya nausar, et nayarin pansengegan na pakaderal na normal ya bituka. Wala ray aromog min pananguman ed karakel na pigaran bakterya, ya mangisusuheri ya say PPA et makaimpluensya ed komposisyon na gut microbiota. Say pananguman ed microbiota et nayarin manresulta ed pananguman ed lebel na pigaran metabolic pathways, ya nayarin manresulta ed pananguman ed pisyolohiya ya makanakana ed bunigas na host. Kaukolan so kaaruman iran panagaral pian naamtaan no kasin say epekto na PPA ed kakanen ed komposisyon na mikrobyo et nayarin pansengegan na dysbiosis odino arum nin sakit. Sayan impanaral so mangiletneg na pundasyon parad arapen iran panaral no panon ya makaapekto ed bunigas na too so epekto na PPA ed komposisyon na bituka.
Saray dataset ya nipresenta ed sayan impanaral et walad saray online repository. Say ngaran na repository tan say access number et: https://www.ncbi.nlm.nih.gov/, PRJNA1092431.
Sayan impanaral ed ayayep et inaprobaan na University of Central Florida Institutional Animal Care and Use Committee (UCF-IACUC) (Animal Use Permit Number: PROTO202000002). Sayan panagaral et mitunosan ed saray lokal a ganggan, totontonen, tan saray kakaukolanen na institusyon.
NG: Konseptuwalisasyon, Data curation, Pormal analysis, Investigation, Methodology, Software, Visualization, Writing (orihinal draft), Writing (review & editing). LA: Konseptuwalisasyon, Data curation, Methodology, Resources, Writing (review & editing). SH: Pormal ya panagsukimat, Software, Pansulat (panrepaso tan panag-edit). SA: Imbestigasyon, Pansulat (panrepaso tan panag-edit). Chief Judge: Imbestigasyon, Pansulat (panrepaso tan panag-edit). SN: Konseptuwalisasyon, Panangasikaso na proyekto, Rekurso, Panangasikaso, Pansulat (panrepaso tan panag-edit). TA: Konseptuwalisasyon, Panangasikaso ed proyekto, Panangasikaso, Pansulat (panrepaso tan panag-edit).
Indeklara na saray autor ya anggapoy naawat dan pinansyal a suporta parad panagsukimat, inka-autor, tan/odino pangipalapag ed sayan artikulo.
Ibabaga na saray autor ya say research et ginawa ya anggapoy antokaman ya komersyal o pinansyal ya relasyon ya nayarin ipasen ya posiblin panlalaban na interes. ag onaplika.
Amin ya opinyon ya nibalikas ed sayan artikulo et parad saray autor labat tan agmet kaukolan ya ipapatnag so opinyon na saray institusyon, publisher, editor, odino reviewer da. Antokaman ya produkto ya inebalwar ed sayan artikulo, odino antokaman ya ibabaga na saray nanggawa ed saratan, et ag-igarantiyaan o inendorso na publisher.
Say kaaruman a materyal parad sayan artikulo et naromog ed online: https://www.frontiersin.org/articles/10.3389/frmbi.2024.1451735/full#supplementary-material
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Oras na post: Apr-18-2025