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Lung cancer (LC) is the most prevalent cancer worldwide, and responsible for over 1.3 million deaths each year. Currently, LC has a low five year survival rates relative to other cancers, and thus, novel methods to screen for and diagnose malignancies are necessary to improve patient outcomes. Here, we report on a pilot-sized study to evaluate the potential of the sputum microbiome as a source of non-invasive bacterial biomarkers for lung cancer status and stage. Spontaneous sputum samples were collected from ten patients referred with possible LC, of which four were eventually diagnosed with LC (LC+), and six had no LC after one year (LC-). Of the seven bacterial species found in all samples, Streptococcus viridans was significantly higher in LC+ samples. Seven further bacterial species were found only in LC-, and 16 were found only in samples from LC+. Additional taxonomic differences were identified in regards to significant fold changes between LC+ and LC-cases, with five species having significantly higher abundances in LC+, with Granulicatella adiacens showing the highest level of abundance change. Functional differences, evident through significant fold changes, included polyamine metabolism and iron siderophore receptors. G. adiacens abundance was correlated with six other bacterial species, namely Enterococcus sp. 130, Streptococcus intermedius, Escherichia coli, S. viridans, Acinetobacter junii, and Streptococcus sp. 6, in LC+ samples only, which could also be related to LC stage. Spontaneous sputum appears to be a viable source of bacterial biomarkers which may have utility as biomarkers for LC status and stage.

Background To address rising demand for dairy products, dairy goats are often fed high-concentrate diets, which lead to subacute rumen acidosis (SARA). The mechanisms behind individual variation in SARA tolerance are not well understood. This study aims to elucidate roles of rumen microbiome-host interactions in SARA-susceptibility and tolerance. Results Goats susceptible or tolerant to SARA were selected by feeding diets with different levels of rumen degradable starch. SARA-susceptible goats present prolonged periods of rumen pH below 5.8 and volatile fatty acids (VFAs) accumulation. Metagenomic analysis reveals a decrease in cellulose- and hemicellulose-utilizing bacteria and enzymes, along with increased lysozymes, suggesting disrupted rumen homeostasis. Transcriptomic and single-nucleus transcriptome analyses reveal upregulated Th17 cells, IL-17 signalling, and inflammatory pathways in SARA-susceptible goats. In contrast, SARA-tolerant goats maintain stable pH levels and enhance VFAs absorption. Bifidobacterium adolescentis and other beneficial bacteria are enriched in the rumen of SARA-tolerant goats. These microbes are positively correlated with 3-methyl pyruvic acid, a key metabolite involved in branched-chain amino acid synthesis and epithelial cell proliferation. Both microbiome transplantation and B. adolescentis direct feeding experiments confirm the protective effects of SARA-tolerant microbiota including B. adolescentis , promoting rumen epithelial VFAs absorption and reducing ruminal inflammation. Conclusions This study highlights the importance of Th17-mediated immune responses in ruminal inflammation and the role of B. adolescentis in regulating rumen epithelial VFAs absorption. Modulating VFAs absorption in the rumen epithelium represents a promising strategy for improving animal health and enhancing rumen fermentation efficiency.

Background The rumen contains a myriad of microbes whose primary role is to degrade and ferment dietary nutrients, which then provide the host with energy and nutrients. Rumen microbes commonly attach to ingested plant materials and form biofilms for effective plant degradation. Quorum sensing (QS) is a well-recognised form of bacterial communication in most biofilm communities, with homoserine lactone (AHL)-based QS commonly being used by Gram-negative bacteria alone and AI-2 Lux-based QS communication being used to communicate across Gram-negative and Gram-positive bacteria. However, bacterial cell to cell communication in the rumen is poorly understood. In this study, rumen bacterial genomes from the Hungate collection and Genbank were prospected for QS-related genes. To check that the discovered QS genes are actually expressed in the rumen, we investigated expression levels in rumen metatranscriptome datasets. Results A total of 448 rumen bacterial genomes from the Hungate collection and Genbank, comprised of 311 Gram-positive, 136 Gram-negative and 1 Gram stain variable bacterium, were analysed. Abundance and distribution of AHL and AI-2 signalling genes showed that only one species ( Citrobacter sp. NLAE-zl-C269) of a Gram-negative bacteria appeared to possess an AHL synthase gene, while the Lux-based genes (AI-2 QS) were identified in both Gram-positive and Gram-positive bacteria (191 genomes representing 38.2% of total genomes). Of these 192 genomes, 139 are from Gram-positive bactreetteria and 53 from Gram-negative bacteria. We also found that the genera Butyrivibrio , Prevotella , Ruminococcus and Pseudobutyrivibrio , which are well known as the most abundant bacterial genera in the rumen, possessed the most lux-based AI-2 QS genes. Gene expression levels within the metatranscriptome dataset showed that Prevotella , in particular, expressed high levels of LuxS synthase suggesting that this genus plays an important role in QS within the rumen. Conclusion This is the most comprehensive study of QS in the rumen microbiome to date. This study shows that AI-2-based QS is rife in the rumen. These results allow a greater understanding on plant-microbe interactions in the rumen.

Antimicrobial Peptides (AMPs) are key constituents of the invertebrate innate immune system and provide critical protection against microbial threat. Nematodes display diverse life strategies where they are exposed to heterogenous, microbe rich, environments highlighting their need for an innate immune system. Within the Ecdysozoa, arthropod AMPs have been well characterised, however nematode-derived AMP knowledge is limited. In this study the distribution and abundance of putative AMP-encoding genes was examined in 134 nematode genomes providing the most comprehensive profile of AMP candidates within phylum Nematoda. Through genome and transcriptome analyses we reveal that phylum Nematoda is a rich source of putative AMP diversity and demonstrate (i) putative AMP group profiles that are influenced by nematode lifestyle where free-living nematodes appear to display enriched putative AMP profiles relative to parasitic species; (ii) major differences in the putative AMP profiles between nematode clades where Clade 9/V and 10/IV species possess expanded putative AMP repertoires; (iii) AMP groups with highly restricted profiles (e.g. Cecropins and Diapausins) and others [e.g. Nemapores and Glycine Rich Secreted Peptides (GRSPs)] which are more widely distributed; (iv) complexity in the distribution and abundance of CSαβ subgroup members; and (v) that putative AMPs are expressed in host-facing life stages and biofluids of key nematode parasites. These data indicate that phylum Nematoda displays diversity in putative AMPs and underscores the need for functional characterisation to reveal their role and importance to nematode biology and host-nematode-microbiome interactions.

Background Liver damage from nonalcoholic steatohepatitis (NASH) presents a significant challenge to the health and productivity of ruminants. However, the regulatory mechanisms behind variations in NASH susceptibility remain unclear. The gut‒liver axis, particularly the enterohepatic circulation of bile acids (BAs), plays a crucial role in regulating the liver diseases. Since the ileum is the primary site for BAs reabsorption and return to the liver, we analysed the ileal metagenome and metabolome, liver and serum metabolome, and liver single-nuclei transcriptome of NASH-resistant and susceptible goats together with a mice validation model to explore how ileal microbial BAs metabolism affects liver metabolism and immunity, uncovering the key mechanisms behind varied NASH pathogenesis in dairy goats. Results In NASH goats, increased total cholesterol (TC), triglyceride (TG), and primary BAs and decreased secondary BAs in the liver and serum promoted hepatic fat accumulation. Increased ileal Escherichia coli , Erysipelotrichaceae bacterium and Streptococcus pneumoniae as well as proinflammatory compounds damaged ileal histological morphology, and increased ileal permeability contributes to liver inflammation. In NASH-tolerance (NASH-T) goats, increased ursodeoxycholic acid (UDCA), isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) in the liver, serum and ileal contents were attributed to ileal secondary BAs-producing bacteria ( Clostridium , Bifidobacterium and Lactobacillus ) and key microbial genes encoding enzymes. Meanwhile, decreased T-helper 17 (T H 17) cells and increased regulatory T (T reg ) cells proportion were identified in both liver and ileum of NASH-T goats. To further validate whether these key BAs affected the progression of NASH by regulating the proliferation of T H 17 and T reg cells, the oral administration of bacterial UDCA, isoDCA and isoLCA to a high-fat diet-induced NASH mouse model confirmed the amelioration of NASH through the T H 17 cell differentiation/IL-17 signalling/PPAR signalling pathway by these bacterial secondary BAs. Conclusion This study revealed the roles of ileal microbiome and its secondary BAs in resilience and susceptibility to NASH by affecting the hepatic T reg and T H 17 cells proportion in dairy goats. Bacterial UDCA, isoDCA and isoLCA were demonstrated to alleviate NASH and could be novel postbiotics to modulate and improve the liver health in ruminants. A8SM95maBgMZc1-WqV_tnD Video Abstract

Background Despite the growing number of studies investigating the connection between host genetics and the rumen microbiota, there remains a dearth of systematic research exploring the composition, function, and metabolic traits of highly heritable rumen microbiota influenced by host genetics. Furthermore, the impact of these highly heritable subsets on lactation performance in cows remains unknown. To address this gap, we collected and analyzed whole-genome resequencing data, rumen metagenomes, rumen metabolomes and short-chain fatty acids (SCFAs) content, and lactation performance phenotypes from a cohort of 304 dairy cows. Results The results indicated that the proportions of highly heritable subsets ( h 2  ≥ 0.2) of the rumen microbial composition (55%), function (39% KEGG and 28% CAZy), and metabolites (18%) decreased sequentially. Moreover, the highly heritable microbes can increase energy-corrected milk (ECM) production by reducing the rumen acetate/propionate ratio, according to the structural equation model (SEM) analysis (CFI = 0.898). Furthermore, the highly heritable enzymes involved in the SCFA synthesis metabolic pathway can promote the synthesis of propionate and inhibit the acetate synthesis. Next, the same significant SNP variants were used to integrate information from genome-wide association studies (GWASs), microbiome-GWASs, metabolome-GWASs, and microbiome-wide association studies (mWASs). The identified single nucleotide polymorphisms (SNPs) of rs43470227 and rs43472732 on SLC30A9 (Zn 2+ transport) ( P  < 0.05/nSNPs) can affect the abundance of rumen microbes such as Prevotella_sp. , Prevotella_sp._E15-22 , Prevotella_sp._E13-27 , which have the oligosaccharide-degradation enzymes genes, including the GH10, GH13, GH43, GH95, and GH115 families. The identified SNPs of chr25:11,177 on 5s_rRNA (small ribosomal RNA) ( P  < 0.05/nSNPs) were linked to ECM, the abundance alteration of Pseudobutyrivibrio_sp. (a genus that was also showed to be linked to the ECM production via the mWASs analysis), GH24 (lysozyme), and 9,10,13-TriHOME (linoleic acid metabolism). Moreover, ECM, and the abundances of Pseudobutyrivibrio sp., GH24, and 9,10,13-TRIHOME were significantly greater in the GG genotype than in the AG genotype at chr25:11,177 ( P  < 0.05). By further the SEM analysis, GH24 was positively correlated with Pseudobutyrivibrio sp . , which was positively correlated with 9,10,13-triHOME and subsequently positively correlated with ECM (CFI = 0.942). Conclusion Our comprehensive study revealed the distinct heritability patterns of rumen microbial composition, function, and metabolism. Additionally, we shed light on the influence of host SNP variants on the rumen microbes with carbohydrate metabolism and their subsequent effects on lactation performance. Collectively, these findings offer compelling evidence for the host-microbe interactions, wherein cows actively modulate their rumen microbiota through SNP variants to regulate their own lactation performance. C1VA69ck9huqCRa2wNrTKW Video Abstract

Antimicrobial Peptides (AMPs) are immune effectors that are key components of the invertebrate innate immune system providing protection against pathogenic microbes. Parasitic helminths (phylum Nematoda and phylum Platyhelminthes) share complex interactions with their hosts and closely associated microbiota that are likely regulated by a diverse portfolio of antimicrobial immune effectors including AMPs. Knowledge of helminth AMPs has largely been derived from nematodes, whereas the flatworm AMP repertoire has not been described. This study highlights limitations in the homology-based approaches, used to identify putative nematode AMPs, for the characterisation of flatworm AMPs, and reveals that innovative algorithmic AMP prediction approaches provide an alternative strategy for novel helminth AMP discovery. The data presented here: (i) reveal that flatworms do not encode traditional lophotrochozoan AMP groups (Big Defensin, CSαβ peptides and Myticalin); (ii) describe a unique integrated computational pipeline for the discovery of novel helminth AMPs; (iii) reveal >16,000 putative AMP-like peptides across 127 helminth species; (iv) highlight that cysteine-rich peptides dominate helminth AMP-like peptide profiles; (v) uncover eight novel helminth AMP-like peptides with diverse antibacterial activities, and (vi) demonstrate the detection of AMP-like peptides from Ascaris suum biofluid. These data represent a significant advance in our understanding of the putative helminth AMP repertoire and underscore a potential untapped source of antimicrobial diversity which may provide opportunities for the discovery of novel antimicrobials. Further, unravelling the role of endogenous worm-derived antimicrobials and their potential to influence host-worm-microbiome interactions may be exploited for the development of unique helminth control approaches.

Background Ruminant products are widely consumed due to their high protein and micronutrient content, but ruminant production contributes significantly to greenhouse gas emissions, with methane (CH₄) accounting for 33% of anthropogenic emissions. CH₄ is generated via fermentative processes by the rumen microbiome, primarily through hydrogen utilisation by methanogenic archaea. Feeding beef cattle the red seaweed Asparagopsis taxiformis (ASP) has been shown to reduce CH₄ emissions by up to 80%. However, the microbial mechanisms underlying this reduction remain poorly understood. In this study, Nordic Red dairy cows (122 ± 13.7 days in milk) were fed grass silage and concentrate (60:40 dry matter basis) either with or without 0.5% ASP (organic matter basis) in a Latin square design, and rumen fluid was collected 19 days into each of the 3 experimental periods. Results ASP supplementation reduced CH₄ yield by 54% (g CH₄/kg DM). Metagenomic analysis revealed genes encoding pyruvate and propionate production pathways were more abundant in ASP treated animals, while those associated with acetate and CH₄ were reduced. Additionally, genes encoding vitamin B12 biosynthesis enzymes showed reduced abundances (e.g., adenosylcobinamide-GDP ribazoletransferase, EC 2.7.8.26, −29.92%). Vitamin B12 and its related cofactors are critical for methanogenic methyltransferases and C1 metabolism. Dominant taxa including Prevotella and Methanobrevibacter declined, while less abundant taxa increased their contribution to methane-related pathways, indicating niche displacement and community restructuring. Conclusion  ASP supplementation modulates the rumen microbiome through mechanisms extending beyond direct methanogen inhibition. The reduced abundance of genes involved in C1 metabolism and vitamin B12-dependent methanogenic processes suggest methane suppression is linked to broader restructuring of microbial metabolic networks. The redistribution of methane-related functions from dominant taxa to a wider taxonomic community indicates ecological reorganisation and functional resilience of the rumen microbiome. Collectively, these results reveal the multiple modes of action of ASP, establishing its promise as an effective methane mitigation strategy. 4PYchXenjeLoFb7wmTFg24 Video Abstract

The temporal variability of the human microbiome may be an important factor in determining its relationship with health and disease. In this study, the saliva of 40 participants was collected every 2 months over a one-year period to determine the temporal variability of the human salivary microbiome. Salivary pH and 16S rRNA gene copy number were measured for all participants, with the microbiome of 10 participants assessed through 16S rRNA amplicon sequencing. In February 2013, 16S rRNA gene copy number was significantly (P < 0.001) higher, with individual changes between time points significant (P = 0.003). Salivary pH levels were significantly (P < 0.001) higher in December 2012 than in October 2012 and February 2013, with significant (P < 0.001) individual variations seen throughout. Bacterial α-diversity showed significant differences between participants (P < 0.001), but not sampling periods (P = 0.801), and a significant positive correlation with salivary pH (R2 = 7.8%; P = 0.019). At the phylum level, significant differences were evident between participants in the Actinobacteria (P < 0.001), Bacteroidetes (P < 0.001), Firmicutes (P = 0.008), Fusobacteria (P < 0.001), Proteobacteria (P < 0.001), Synergistetes (P < 0.001) and Spirochaetes (P = 0.003) phyla. This study charted the temporal variability of the salivary microbiome, suggesting that bacterial diversity is stable, but that 16S rRNA gene copy number may be subject to seasonal flux. This is the first study to chart the temporal variability of the salivary microbiome and suggested that this was stable in terms of its diversity but not load over a one-year period.

Eicosapentaenoic acid (EPA) from freeze-dried biomass of Nannochloropsis oceanica microalgae resists ruminal biohydrogenation in vitro, but in vivo demonstration is needed. Therefore, the present study was designed to test the rumen protective effects of N. oceanica in lambs. Twenty-eight lambs were assigned to one of four diets: Control (C); and C diets supplemented with: 1.2% Nannochloropsis sp. oil (O); 12.3% spray-dried N. oceanica (SD); or 9.2% N. oceanica (FD), to achieve 3 g EPA /kg dry matter. Lambs were slaughtered after 3 weeks and digestive contents and ruminal wall samples were collected. EPA concentration in the rumen of lambs fed FD was about 50% higher than lambs fed SD or O diets. Nevertheless, the high levels of EPA in cecum and faeces of animals fed N. oceanica biomass, independently of the drying method, suggests that EPA was not completely released and absorbed in the small intestine. Furthermore, supplementation with EPA sources also affected the ruminal biohydrogenation of C18 fatty acids, mitigating the shift from the t 10 biohydrogenation pathways to the t 11 pathways compared to the Control diet. Overall, our results demonstrate that FD N. oceanica biomass is a natural rumen-protected source of EPA to ruminants.