Alterations in bile acid profile and pathways contribute to hepatic inflammation in cancer cachexia, a syndrome worsening the prognosis of cancer patients. As the gut microbiota impinges on host metabolism through bile acids, the current study aimed to explore the functional contribution of gut microbial dysbiosis to bile acid dysmetabolism and associated disorders in cancer cachexia. Using three mouse models of cancer cachexia (the C26, MC38 and HCT116 models), we evidenced a reduction in the hepatic levels of several secondary bile acids, mainly taurodeoxycholic (TDCA). This reduction in hepatic TDCA occurred before the appearance of cachexia. Longitudinal analysis of the gut microbiota pinpointed an ASV, identified as Xylanibacter rodentium, as a bacterium potentially involved in the reduced production of TDCA. Coherently, stable isotope-based experiments highlighted a robust decrease in the microbial 7α-dehydroxylation (7α-DH) activity with no changes in the bile salt hydrolase (BSH) activity in cachectic mice. This approach also highlighted a reduced microbial 7α-hydroxysteroid dehydrogenase (7α-HSDH) and 12α-hydroxysteroid dehydrogenase (12α-HSDH) activities in these mice. The contribution of the lower production of TDCA to cancer cachexia was explored in vitro and in vivo. In vitro, TDCA prevented myotube atrophy, whereas in vivo hepatic whole transcriptome analysis revealed that TDCA administration to cachectic mice improved the unfolded protein response and cholesterol homeostasis pathways. Coherently, TDCA administration reversed hepatic cholesterol accumulation in these mice. Altogether, this work highlights the contribution of the gut microbiota to bile acid dysmetabolism and the therapeutic interest of the secondary bile acid TDCA for hepatic cholesterol homeostasis in the context of cancer cachexia. Such discovery may prove instrumental in the understanding of other metabolic diseases characterized by microbial dysbiosis. More broadly, our work demonstrates the interest and relevance of microbial activity measurements using stable isotopes, an approach currently underused in the microbiome field.

N6-methyladenosine (m6A) is an important epigenetic modification in RNA, playing a crucial role in regulating the production and aging of animal testicular sperm. This study extracted mRNA from the testicular tissue of male goats before and after sexual maturity, generating a methylation map through preliminary experiments and methylation immunoprecipitation sequencing. The results showed that during the development of dairy goats, the expression levels of marker genes related to testicular development and methylation-related enzymes changed significantly. A total of 36,602 peaks and 11,223 genes were identified in the two groups, including 2989 differential peaks (427 upregulated and 2562 downregulated) and 1457 differentially expressed genes (833 upregulated and 624 downregulated). The abundance of m6A was positively correlated with gene expression levels. This study reports for the first time the mRNA profiles of m6A modifications across the entire transcriptome during testicular development in Guanzhong dairy goats, providing a new perspective for genetic improvement in goats.

Crohn's disease (CD) and ulcerative colitis (UC) are chronic relapsing inflammatory bowel disorders (IBD), the pathogenesis of which is uncertain but includes genetic susceptibility factors, immune-mediated tissue injury and environmental influences, most of which appear to act via the gut microbiome. We hypothesized that host-microbe alterations could be used to prognostically stratify patients experiencing relapses up to four years after endoscopy. We therefore examined multiple omics data, including published and new datasets, generated from paired inflamed and non-inflamed mucosal biopsies from 142 patients with IBD (54 CD; 88 UC) and from 34 control (non-diseased) biopsies. The relapse-predictive potential of 16S rRNA gene and transcript amplicons (standing and active microbiota) were investigated along with host transcriptomics, epigenomics and genetics. While standard single-omics analysis could not distinguish between patients who relapsed and those that remained in remission within four years of colonoscopy, we did find an association between the number of flares and a patient's succinotype. Our multi-omics machine learning approach was also able to predict relapse when combining features from the microbiome and human host. Therefore multi-omics, rather than single omics, better predicts relapse within 4 years of colonoscopy, while a patient's succinotype is associated with a higher frequency of relapses.

Serratia marcescens (S. marcescens) commonly induces refractory infection due to its multidrug-resistant nature. To date, there have been no reports on the application of phage treatment for S. marcescens infection. This study was conducted to explore the feasibility of phage application in treating refractory S. marcescens infection by collaborating with a 59-year-old male patient with a pulmonary infection of multidrug-resistant S. marcescens. Our experiments included three domains: i) selection of the appropriate phage, ii) verification of the efficacy and safety of the selected phage, iii) confirmation of phage-bacteria interactions. Our results showed that phage Spe5P4 is appropriate for S. marcescens infection. Treatment with phage Spe5P4 showed good efficacy, manifested as amelioration of symptoms, hydrothorax examinations, and chest computed tomography findings. Phage treatment did not worsen hepatic and renal function, immunity-related indices, or indices of routine blood examination. It did not induce or deteriorate drug resistance of the involved antibiotics. Importantly, no adverse events were reported during the treatment or follow-up periods. Thus, phage treatment showed satisfactory safety. Finally, we found that phage treatment did not increase the bacterial load, cytotoxicity, virulence, or phage resistance of S. marcescens, indicating satisfactory phage-bacteria interactions between Spe5P4 and S. marcescens, which are useful for the future application of phage Spe5P4 against S. marcescens. This work provides evidence and a working basis for further application of phage Spe5P4 in treating refractory S. marcescens infections. We also provided a methodological basis for investigating clinical application of phage treatment against multidrug-resistant bacterial infections in the future.

An expansion of plasma anelloviruses and dysregulation of inflammation was associated with HIV-1 infection. However, how antiretroviral therapy (ART) affects the dynamics of plasma virome and cytokine profile remains largely unknown. To characterize the dynamics of plasma virome and cytokines in HIV-1-infected individuals before and during the first year of ART, a cohort of 26 HIV-1-infected individuals and 19 healthy controls was recruited. Blood samples were collected and subjected to metagenomic analysis and the measurement of 27 cytokines. Metagenomic analysis revealed an increased abundance and prevalence of human pegivirus type 1 (HPgV-1) and a slightly decreased diversity and abundance of anellovirus in plasma of HIV-1-infected individuals after ART. No obvious impact was observed on other plasma commensal viruses. Increased abundance and prevalence of HPgV-1 were further confirmed by RT-qPCR assay in a larger cohort of 114 HIV-1-infected individuals. Notably, most dysregulated cytokines were not fully restored by ART, with extremely abnormal levels of IL-10, GM-CSF, VEGF, and eotaxin, and a significantly increased level of plasma I-FABP. Anelloviruses showed significantly negative correlations with other commensal viruses except HPgV-1 but had positive correlations with several anti-inflammatory and Th1 cytokines. These results suggest that short-term ART may not significantly correct the virome and cytokine dysregulations induced by HIV-1 infection. The results highlight a need for further investigation into the long-term effects of ART on virome and cytokine profiles in HIV-1-infected individuals.

Bacterial genomic DNA inversions, which govern molecular phase-variations, provide the bacteria with functional plasticity and phenotypic diversity. These targeted rearrangements enable bacteria to respond to environmental challenges, such as bacteriophage predation, evading immune detection or gut colonization. This study investigated the short- and long-term effects of the lytic phage Barc2635 on the functional plasticity of Bacteroides fragilis, a gut commensal. Germ-free mice were colonized with B. fragilis and exposed to Barc2635 to identify genomic alterations driving phenotypic changes. Phage exposure triggered dynamic and prolonged bacterial responses, including significant shifts in phase-variable regions (PVRs), particularly in promoter orientations of polysaccharide biosynthesis loci. These shifts coincided with increased entropy in PVR inversion ratios, reflecting heightened genomic variability. In contrast, B. fragilis in control mice exhibited stable genomic configurations after gut adaptation. The phase-variable Type 1 restriction-modification system, which affects broad gene expression patterns, showed variability in both groups. However, phage-exposed bacteria displayed more restrained variability, suggesting phage-derived selection pressures. Our findings reveal that B. fragilis employs DNA inversions to adapt rapidly to phage exposure and colonization, highlighting a potential mechanism by which genomic variability contributes to its response to phage. This study demonstrates gut bacterial genomic and phenotypic plasticity upon exposure to the mammalian host and to bacteriophages.

Reliable engraftment assessment of donor microbial communities and individual strains is an essential component of characterizing the pharmacokinetics of microbiota transplant therapies (MTTs). Recent methods for measuring donor engraftment use whole-genome sequencing and reference databases or metagenome-assembled genomes (MAGs) to track individual bacterial strains but lack the ability to disambiguate DNA that matches both donor and patient microbiota. Here, we describe a new, cost-efficient analytic pipeline, MAGEnTa, which compares post-MTT samples to a database comprised MAGs derived directly from donor and pre-treatment metagenomic data, without relying on an external database. The pipeline uses Bayesian statistics to determine the likely sources of ambiguous reads that align with both the donor and pre-treatment samples. MAGEnTa recovers engrafted strains with minimal type II error in a simulated dataset and is robust to shallow sequencing depths in a downsampled dataset. Applying MAGEnTa to a dataset from a recent MTT clinical trial for ulcerative colitis, we found the results to be consistent with 16S rRNA gene SourceTracker analysis but with added MAG-level specificity. MAGEnTa is a powerful tool to study community and strain engraftment dynamics in the development of MTT-based treatments that can be integrated into frameworks for functional and taxonomic analysis.

The emergence of multidrug-resistance (MDR) in Pasteurella multocida, a major contributor to bovine respiratory disease (BRD) is being increasingly reported, often linked to the carriage of antimicrobial resistance genes (ARGs) on integrative and conjugative elements (ICEs). The resistance phenotype for 19 antimicrobials was determined using broth microdilution in 75 Pasteurella multocida isolates from healthy and BRD-affected cattle from five feedlots. The genomes of 32 isolates were sequenced to identify ARG) and mobile genetic elements (MGEs) and assess their genetic diversity. MDR isolates (with phenotypic resistance to aminoglycosides, macrolides, fluoroquinolones and/or tetracyclines) were primarily found among BRD-affected compared to healthy animals. Non-susceptible isolates, belonging to ST79 and ST13, harbored point mutations and four to nine ARGs, including rarely reported mechanisms in Europe (mph(E), msr(E) and aadA31 ARGs and newly described mutations in the gyrA/parC genes). All ARGs were linked to the presence of MGEs including two ICEs, Tn7407 and the novel Tn7809, a prophage and a putative composite transposon. Clonally related isolates were found in different batches from the same feedlot, suggesting maintenance of MDR strains. Our findings demonstrate the diverse genetic basis of AMR in P. multocida from BRD-affected cattle in Spain, emphasizing the role of MGEs in the ARG dissemination.

The role of xenobiotic disruption of microbiota, corresponding dysbiosis, and potential links to host metabolic diseases are of critical importance. In this study, we used a widely prescribed antipsychotic drug, risperidone, known to influence weight gain in humans, to induce weight gain in C57BL/6J female mice. We hypothesized that microbes essential for maintaining gut homeostasis and energy balance would be depleted following treatment with risperidone, leading to enhanced weight gain relative to controls. Thus, we performed metagenomic analyses on stool samples to identify microbes that were excluded in risperidone-treated animals but remained present in controls. We identified multiple taxa including Limosilactobacillus reuteri as a candidate for further study. Oral supplementation with L. reuteri protected against risperidone-induced weight gain (RIWG) and was dependent on cellular production of a specialized metabolite, reutericyclin. Further, synthetic reutericyclin was sufficient to mitigate RIWG. Both synthetic reutericyclin and L. reuteri restored energy balance in the presence of risperidone to mitigate excess weight gain and induce shifts in the microbiome associated with leanness. In total, our results identify reutericyclin production by L. reuteri as a potential probiotic to restore energy balance induced by risperidone and to promote leanness.

Necrotizing enterocolitis (NEC) remains a frequent catastrophic disease in preterm infants, and fecal filtrate transfer (FFT) has emerged as a promising prophylactic therapy. This study explored the role of virome viability for the protective effect of FFT. Using ultraviolet (UV) irradiation, we established a viral inactivation protocol and administered FFT, UV-inactivated FFT (iFFT) or sterile saline orally to preterm piglets at risk for experimental NEC. The gut pathology and barrier properties were assessed, while the microbiome was explored by 16S rRNA amplicon and metavirome sequencing. Like in prior studies, FFT reduced NEC severity and intestinal inflammation, while these effects were absent in the iFFT group. Unexpectedly, piglets receiving FFT exhibited mild side effects in the form of early-onset diarrhea. The FFT also converged the gut colonization by increased viral heterogeneity and a reduced abundance of pathobionts like Clostridium perfringens and Escherichia. In contrast, the gut microbiome of iFFT recipients diverged from both FFT and the controls. These findings highlight the clear distinction between the ability of active and inactivate viromes to modulate gut microbiota and decrease pathology. The efficacy of FFT may be driven by active bacteriophages, and loss of virome activity could have consequences for the treatment efficacy.

Rhizosphere bacterial community studies offer valuable insights into the environmental implications of genetically modified (GM) crops. This study compared the effects of a non-GM maize cultivar, namely Hi-IIA, with those of a herbicide-resistant maize cultivar containing the phosphinothricin N-acetyltransferase gene on the rhizosphere bacterial community across growth stages. 16s rRNA amplicon sequencing and data analysis tools revealed no significant differences in bacterial community composition or diversity between the cultivars. Principal component analysis revealed that differences in community structure were driven by plant growth stages rather than plant type. Polymerase chain reaction analysis was conducted to examine the potential horizontal transfer of the introduced gene from the GM maize to rhizosphere microorganisms; however, the introduced gene was not detected in the soil genomic DNA. Overall, the environmental impact of GM maize, particularly on soil microorganisms, is negligible, and the cultivation of GM maize does not alter significantly the rhizosphere bacterial community.

Honey bees are important pollinators in both agriculture and ecosystems, and their health is essential for sustainable human development. Although only two bacteria, Paenibacillus larvae and Melissococcus plutonius, have been identified as bacterial pathogens in honey bee brood for over 100 years, we found three additional Paenibacillus strains (Paenibacillus sp. J27TS7, Paenibacillus azoreducens J34TS1, and Paenibacillus melissococcoides J46TS7) in honey that harmed honey bee brood development. In particular, Paenibacillus sp. J27TS7 was highly virulent in bee larvae (the median lethal dose [LD50] = 12.7 spores/larva) and was comparable to P. larvae (LD50 = 2.3-11.5 spores/larva). Paenibacillus azoreducens J34TS1 showed the second-highest virulence (LD50 = 45.9 spores/larva), and P. melissococcoides J46TS7 was the least virulent (LD50 = 469.0 spores/larva). However, P. melissococcoides was most frequently detected in Japanese honey among the three species, with the highest concentration being 1.8 × 106 spores/mL honey, suggesting its wide distribution in Japanese apiaries. The novel pathogenic Paenibacillus species were categorized into the fast killer (Paenibacillus sp. J27TS7), medium-fast killer (P. melissococcoides), and slow killer (P. azoreducens) like P. larvae strains in terms of the time to kill infected brood; however, histopathological and genome analyses indicated that their pathogenic mechanisms were different from those of P. larvae strains. Moreover, P. melissococcoides showed differences in virulence depending on the lineage of the strain. These findings represent the first discovery of honey bee brood pathogens in more than 100 years and indicate the need to look beyond known pathogens for a comprehensive understanding of honey bee diseases.

Super- and low-shedding phenomena have been observed in genetically homogeneous hosts infected by a single bacterial strain. To decipher the mechanisms underlying these phenotypes, we conducted an experiment with chicks infected with Salmonella Enteritidis in a non-sterile isolator, which prevents bacterial transmission between animals while allowing the development of the gut microbiota. We investigated the impact of four commensal bacteria called Mix4, inoculated at hatching, on chicken systemic immune response and intestinal microbiota composition and functions, before and after Salmonella infection. Our results revealed that these phenotypes were not linked to changes in cell invasion capacity of bacteria during infection. Mix4 inoculation had both short- and long-term effects on immune response and microbiota and promoted the low-shedder phenotype. Kinetic analysis revealed that Mix4 activated immune response from day 4, which modified the microbiota on day 6. This change promotes a more fermentative microbiota, using the aromatic compounds degradation pathway, which inhibited Salmonella colonization by day 11 and beyond. In contrast, control animals exhibited a delayed TNF-driven pro-inflammatory response and developed a microbiota using anaerobic respiration, which facilitates Salmonella colonization and growth. This strategy offers promising opportunities to strengthen the barrier effect against Salmonella and possibly other pathogens.

Here, we report that small extracellular vesicles (sEVs) in milk mediate the communication between bacteria and animal kingdoms, increase the divergence of bacteria in the intestine, and alter metabolite production by bacteria. We show that bovine milk sEVs select approximately 55,000 genomic variants in 19 species of bacteria from the murine cecum ex vivo. The genomic variants are transcribed into mRNA. The selection of genomic variants by milk sEVs alters bacterial metabolism, leading to an up to 12-fold difference in the abundance of more than 1000 metabolites in bacteria cultured in milk sEV-free media compared to sEV-containing media. Evidence is particularly strong that selection of genomic variants by milk sEV changes the metabolism of sugars, amino acids, and purines which might contribute to the development of spatial learning and memory deficiencies and seizure phenotypes reported for milk sEV-depleted infants and mice. Human milk is a rich source of sEVs, whereas formula contains only trace amounts of milk sEVs. This report implicates nutritional sEVs in altered microbial metabolism beyond the mere selection of bacterial communities.

The gut microbiota produces short-chain fatty acids (SCFA) and acidifies the proximal colon which inhibits enteric pathogens. However, for many microbiota constituents, how they themselves resist these stresses is unknown. The anaerobic Lachnospiraceae family, which includes the acetogenic genus Blautia, produce SCFA, are genomically diverse, and vary in their capacity to acidify culture media. Here, we investigated how Lachnospiraceae tolerate pH stress and found that subunits of urease were associated with acidification in a random forest model. Urease cleaves urea into ammonia and carbon dioxide, however the role of urease in the physiology of Lachnospiraceae is unknown. We demonstrate that urease-encoding Blautia show urea-dependent changes in SCFA production, acidification, growth, and, strikingly, urease encoding Blautia directly incorporate the carbon from urea into SCFAs. In contrast, ureolytic Klebsiella pneumoniae or Proteus mirabilis do not show the same urea-dependency or carbon salvage. In agreement, the combination of urease and acetogenesis functions is rare in gut taxa. We find that Lachnospiraceae urease and acetogenesis genes can be co-expressed in healthy individuals and colonization of mice with a ureolytic Blautia reduces urea availability in colon contents demonstrating Blautia urease activity in vivo. In human and mouse microbial communities, the acetogenic recycling of urea carbon into acetate by Blautia leads to the incorporation of urea carbon into butyrate indicating carbon salvage into broader metabolite pools. Altogether, this shows that urea plays a central role in the physiology of health-associated Lachnospiraceae which use urea in a distinct manner that is different from that of ureolytic pathogens.

IgA-coated fractions of the intestinal microbiota of Crohn's disease (CD) patients have been shown to contain taxa that hallmark the compositional dysbiosis in CD microbiomes. However, the correlation between other cellular properties of intestinal bacteria and disease has not been explored further, especially for features that are not directly driven by the host immune-system, e.g. the expression of surface sugars by bacteria. By sorting and sequencing IgA-coated and lectin-stained fractions from CD patients microbiota and healthy controls, we found that lectin-stained bacteria were distinct from IgA-coated bacteria, but still displayed specific differences between CD and healthy controls. To exploit the discriminatory potential of both, immunoglobulin coated bacteria and the altered surface sugar expression of bacteria in CD, we developed a multiplexed single cell-based analysis approach for intestinal microbiota. By multi-parameter microbiota flow cytometry (mMFC) we characterized the intestinal microbiota of 55 CD patients and 44 healthy controls for 11-parameters in total, comprising host-immunoglobulin coating and the presence of distinct surface sugar moieties. The data were analyzed by machine-learning to assess disease-specific marker patterns in the microbiota phenotype. mMFC captured detailed characteristics of CD microbiota and identified patterns to classify CD patients. In addition, we identified phenotypic signatures in the CD microbiota which not only reflected remission after 6 weeks of anti-TNF treatment, but were also able to predict remission before the start of an adalimumab treatment course in a pilot study. We here present the proof-of-concept demonstrating that multi-parameter single-cell bacterial phenotyping by mMFC could be a novel tool with high translational potential to expand current microbiome investigations by phenotyping of bacteria to identify disease- and therapy-associated cellular alterations and to reveal novel target properties of bacteria for functional assays and therapeutic approaches.

We performed a comparative study of the non-ribosomal gene content of nucleoli from seven cancer cell lines, using identical methods of purification and analysis. We identified unique chromosomal domains associated with the nucleolus (NADs) and genes within these domains (NAGs). Four cell lines have relatively few NAGs, which appears mostly transcriptionally inactive, consistent with literature. The remaining three lines formed a separate group with nucleoli with unique features and NADS. They constitute larger number of common NAGs, marked by ATAC-seq and having accessible promoters, with histone markers for transcriptional activity and detectable RNA Pol II bound at their promoters. The transcripts of these genes are almost entirely exported from the nucleolus. These results indicate that RNA Pol II dependent transcription in NADs can vary widely in different cell types, presumably dependent on the cell's developmental stage. Nucleolus-associated genes are likely to be distinguished marks reflecting the cell's metabolism.

Breast milk, rich in human milk oligosaccharides (HMOs), supports the early-life colonization of beneficial bacteria such as bifidobacteria and lactobacilli, potentially reducing early-life antibiotic resistance. However, antibiotic treatment may interfere with the beneficial functions of HMO-degrading bacteria. This study investigated the metabolism of HMOs by bifidobacteria and lactobacilli isolated from human milk and mother-infant paired fecal samples, along with their antibiotic resistance profiles. Understanding these species- and sample-type-specific interactions will provide valuable insights into how bioactive components in human milk may shape the infant resistome during early life. A total of 39 Bifidobacterium and 14 Lactobacillaceae strains were isolated from paired mother-infant fecal and breast milk samples. Whole genome sequencing (WGS) allowed functional predictions on the HMO metabolism abilities and the resistance genotype of each strain. In vitro HMO utilization was assessed using growth kinetics assays combined with HMO glycoprofiling in culture supernatant. The minimum inhibitory concentration (MIC) was also determined for each strain. HMO metabolism by the bifidobacteria was species-specific. Bifidobacterium bifidum (B. bifidum) and Bifidobacterium longum subsp. infantis (B. infantis) exhibited the highest capacity for HMO degradation, consistent with genomic predictions. In contrast, lactobacilli were unable to degrade HMOs in vitro but were predicted to metabolize the by-products of HMO degradation. Phenotypic analysis revealed that B. bifidum strains had the lowest levels of antibiotic resistance, while Bifidobacterium animalis subsp. lactis (B. lactis) strains were resistant to most tested antibiotics. Overall, B. bifidum demonstrated the strongest HMO-degrading ability while remaining the most antibiotic-susceptible species. Early-life colonizing bifidobacterial species possess the essential machinery required to degrade HMOs and are highly susceptible to antibiotics. A better understanding of these dynamics could inform clinical strategies to protect and restore the infant gut microbiome, particularly in neonates exposed to antibiotics.

The human gut microbiome, crucial in various diseases, can be utilized to develop diagnostic models through machine learning (ML). The specific tools and parameters used in model construction such as data preprocessing, batch effect removal and modeling algorithms can impact model performance and generalizability. To establish an generally applicable workflow, we divided the ML process into three above-mentioned steps and optimized each sequentially using 83 gut microbiome cohorts across 20 diseases. We tested a total of 156 tool-parameter-algorithm combinations and benchmarked them according to internal- and external- AUCs. At the data preprocessing step, we identified four data preprocessing methods that performed well for regression-type algorithms and one method that excelled for non-regression-type algorithms. At the batch effect removal step, we identified the "ComBat" function from the sva R package as an effective batch effect removal method and compared the performance of various algorithms. Finally, at the ML algorithm selection step, we found that Ridge and Random Forest ranked the best. Our optimized work flow performed similarly comparing with previous exhaustive methods for disease-specific optimizations, thus is generally applicable and can provide a comprehensive guideline for constructing diagnostic models for a range of diseases, potentially serving as a powerful tool for future medical diagnostics.

Previous clinical trials have shown that time-restricted feeding can be involved in regulating the metabolic health of humans and animals. However, the underlying mechanism has not been fully explored. In this study, the pig model was employed to simulate four prevalent human eating habits, with the aim of investigating the impact of gut microbiota and microbial metabolites on gut hormone secretion and appetite regulation. Compared to the ad libitum feeding (ALF) pattern, three time-restricted feeding patterns reduced total food intake and eating time. Meanwhile, three time-restricted feeding patterns induced elevated levels of serum and hypothalamic glucagon-like peptide-1 (GLP-1), while suppressing reward-related circuits in the hypothalamus. It is noteworthy that the early time-restricted feeding (eTRF) pattern increased the number of intestinal enteroendocrine cells (EECs) compared to ALF. Metagenomic and metabonomic analyses revealed that three time-restricted feeding patterns induced colonization of Lactobacillus and significantly increased the levels of its metabolite, indole-3-lactic acid (ILA). Dietary supplementation with ILA exhibited an increasing trend in fasting serum GLP-1 level of piglets. In vitro studies with pig intestinal organoids showed the Lactobacillus metabolite ILA enhanced GLP-1 secretion through the promotion of intestinal stem cell differentiation into EECs, rather than activating the ability of EECs to secrete GLP-1. Overall, time-restricted feeding promoted GLP-1 secretion and affected long-term appetite regulation by promoting the colonization of Lactobacillus and modulating microbial tryptophan metabolism.

Cardiometabolic risk factors, obesity, diabetes and hyperlipidemia contribute to cardiovascular disease (CVD). While platelets are involved in CVD pathogenesis, the relationship between risk factor burden on platelet indices and the platelet transcriptome remains uncertain. Blood was collected from CVD-free adults, measuring platelet count, mean platelet volume (MPV), immature platelet fraction (IPF), and absolute immature platelet fraction (AIPF) by hemogram. Platelets were isolated and analyzed via RNA sequencing. Participants were stratified by number of cardiometabolic risk factors (diabetes, obesity, hyperlipidemia). We calculated median (IQR) values of platelet indices and p-for-trend via linear regression across risk factor burden. To evaluate the association between risk factor burden and platelet transcripts, we performed multivariable linear regression adjusting for age, sex, and race/ethnicity. Among 141 participants, (50.5 ± 14.8 years, 42% male, 26% Black) risk factor burden was associated with increasing platelet size, IPF, and AIPF but not platelet count. Platelet RNA sequencing identified 100 differentially expressed transcripts (p < .01; 66 upregulated, 34 downregulated). Gene ontology enrichment analysis demonstrated upregulated pathways of secondary metabolic processes (NES = 1.96, p < .01), and hematopoietic stem cell proliferation (NES = 1.95, p < .01). Greater cardiometabolic risk factor burden is associated with increased platelet size and immaturity and suggesting novel platelet-mediated mechanisms linking risk factor burden with CVD.

Ulcerative colitis (UC) is an inflammatory bowel disease characterized by recurrent colonic inflammation. Standard treatments focus on controlling inflammation but remain ineffective for one-third of patients. This underscores the need for alternative approaches, such as fecal microbiota transplantation (FMT), which transfers healthy donor microbiota to patients. The role of viruses in this process, however, remains underexplored. To address this, we analyzed the gut virome using metagenomic sequencing of enriched viral particles from 320 longitudinal fecal samples of 44 patients enrolled in the RESTORE-UC FMT trial. Patients were treated with FMTs from healthy donors (allogenic, treatment) or themselves (autologous, control). We found that colonic inflammation, both its presence and location, had a greater impact on the gut virome than FMT itself. In autologous FMT patients, the virome was unstable and showed rapid divergence over time, a phenomenon we termed virome drift. In allogenic FMT patients, the virome temporarily shifted toward the healthy donor, lasting up to 5 weeks and primarily driven by microviruses. Notably, two distinct virome configurations were identified and linked to either healthy donors or patients. In conclusion, inflammation strongly affects the gut virome in UC patients, which may lead to instability and obstruct the engraftment of allogeneic FMT.

Mexiletine (MXT) is the first-line anti-myotonic drug for myotonic dystrophies (MD) and non-dystrophic myotonias (NDM), metabolized by CYP2D6 and CYP1A2 enzymes. We investigated genetic variants in these genes and their influence on MXT response in Italian MD and NDM patients. Fifty patients (MD: 34, NDM: 16) were treated with MXT (200-600 mg daily) for at least two months. Based on the Myotonic Behaviour Scale (MBS) and neurological examination, 37 patients (74 %) were classified as responders (R), while 13 (26 %) were non-responders (NR). Comparison between R and NR revealed associations with 14 genetic variants (12 in CYP2D6, 2 in CYP1A2). In silico analysis suggested eQTL effects on liver and skeletal muscle gene expression. Functional annotation indicated the regulatory roles of these variants. The CYP2D6*2/*41 diplotype showed a nominal association with non-response (OR = 10.8, p = 0.049), being 11 times more frequent in NR (23 %) than in R (3 %). Most common diplotypes (CYP2D6*1/*2, *1/*1, *1/*10, *2/10) corresponded to Normal Metabolizers, while CYP2D6*10/*10, *10/*41, and *41/41 indicated intermediate metabolism, suggesting a higher risk of adverse reactions with concurrent drugs. The CYP2D6*41 allele was more frequent in NR patients than in the European population, supporting its role in MXT response variability. Our findings suggest CYP2D6 and CYP1A2 variants as potential predictors of MXT treatment response in MD and NDM patients.

Bioplastics made of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with a 20 mol% HV fraction are highly desirable in the market for 3-Hydroxyvalerate (HV)-conferred superior thermal, biological, and mechanical properties. Although Haloferax mediterranei (HM) is capable of producing PHBV from food waste, its HV fraction is generally lower than 10 mol%. This study for the first time investigated the engineering approach to increasing HV fraction through elevating the propionic and valeric acid fractions in volatile fatty acids (VFAs) produced from food waste via arrested anaerobic digestion with and without microbial electrolysis cells (MECs) incorporation. Results showed that HV fraction in PHBV produced by HM is proportional to the fractions of propionic and valeric acids in VFAs. A 20 mol% HV fraction can be achieved by MECs incorporation, which might be attributable to pH regulation by the MECs. These findings lay a foundation for developing waste-processing technologies that enable the production of high-value, microbially-derived materials.

In this study, we prepared a series of composites with various chitosan-to-starch ratios by means of screw extrusion. The morphology and structural order of the composite were examined, and the fermentability was investigated through an in vitro human fecal fermentation model. Fourier transform infrared (FTIR) spectroscopy demonstrated the formation of hydrogen bonds between starch and chitosan in the composite, and X-ray diffraction (XRD) analysis indicated the structural order of the composite was enhanced with increasing chitosan proportions. The addition of chitosan slowed down the pH drop rate during the fermentation, and high chitosan proportion decreased the production of short-chain fatty acids (SCFAs), especially butyrate. The final butyrate concentrations of the composite with chitosan to starch ratio of 1:6 (CS:S = 1:6, 0.97 mmol/g) was almost twice that of the composite with chitosan to starch ratio of 2:1 (CS:S = 2:1, 0.51 mmol/g). Health-promoting microbes including Agathobacter, Prevotella_9, Ruminococcus, and Bifidobacterium were obviously promoted by extruded starch. Moreover, the composite sample with chitosan to starch ratio of 1:6 (CS:S = 1:6) exhibited a good synergistic effect in regulating the microbiota composition, reflected by the increase in the relative abundance of Agathobacter and Prevotella_9. This finding demonstrated that the ratio of starch to chitosan might be a controlling factor in determining the fermentation properties of screw extruded composite.

Oocyte maturation is a critical process for successful fertilization and early embryonic development. In this study, we investigated the molecular mechanisms underlying oocyte maturation in dogs by analyzing the transcriptomic profiles of miRNAs and mRNAs in canine cumulus cells (CCs) and oocytes during in vitro maturation (IVM). RNA sequencing identified 285 miRNAs expressed in oocytes and 310 in CCs, with 282 miRNAs shared between the two cell types, highlighting the role of intercellular communication in maintaining miRNA expression equilibrium. Differential expression analysis revealed 222 mRNAs with significant differences between CCs and oocytes, including genes involved in transcriptional regulation and nuclear structure. Enrichment analyses identified pathways such as actin cytoskeleton regulation, mTOR signaling, cAMP signaling, and calcium signaling, all critical to oocyte maturation. Network analysis revealed 643 significant miRNA-mRNA coexpression relationships, suggesting miRNAs play pivotal roles in regulating mRNA expression during oocyte maturation. Notably, key miRNAs such as miR-30b, miR-375, and miR-503 were implicated in regulating genes involved in oocyte maturation pathways, while others like miR-378 and miR-21 aligned with known roles in suppressing cumulus expansion and influencing maturation. The absence of differential miRNA expression between CCs and oocytes suggests the miRNA transfer through gap junctions. These findings provide new insights into the transcriptional and post-transcriptional regulation of oocyte maturation in dogs, offering valuable knowledge to improve reproductive biotechnologies such as in vitro fertilization and embryo development in this species.

The intramuscular fat (IMF), fatty acid and amino acid compositions of pork are intricately linked to meat quality, flavor profile, and nutritional composition, and have potential implications for human health. Lipid accumulation in pork is initiated by the biosynthesis of fatty acids and regulated by a complex network of genes. In this study, the IMF content and genotyping of large-scale slaughtered Yorkshire pigs were assessed. Transcriptome sequencing of muscles from 17 individuals and fatty and amino acid analyses of muscles from 28 individuals according to IMF content were conducted. Phenotypic analysis showed a high correlation between IMF and most fatty acids, and the composition ratio of different types of fatty acids varied with IMF content. A negative correlation between the n-6/n-3 polyunsaturated fatty acid (PUFA) ratio and increase in IMF content significantly enhanced the levels of essential fatty acids and ameliorated the n-6/n-3 PUFA ratio in pork, thereby elevating its nutritional value to better align with contemporary health standards. A comprehensive analysis that integrated a genome-wide association study, differential gene expression analysis, and weighted gene co-expression network analysis was employed to identify the regulatory mechanisms of lipids. PRLR, SEC11C, ALPK2, CPLX4, APC, and CREB5 were identified as key candidate genes that affect intramuscular lipids and fatty acids. Through molecular and cellular experiments, our results indicated that high APC and CREB5 gene expression significantly promotes lipogenesis in cells, where these genes play an important role in regulatory pathways related to lipid synthesis in animals, which may affect fat deposition and fatty acid composition in pork. Overall, these results lay the foundation for an in-depth analysis of the genetic regulation of pork lipids and nutrition, and also provide molecular regulatory markers for the primary selection of pigs with better meat quality.

This study investigated a novel thermal-hydrolysis combined with a vacuum fermentation system for high-grade volatile fatty acids (VFA) recovery, and the corresponding changes in the microbial community. Four systems with and without hydrothermal pre-treatment (HTP) and vacuum were mobilized; results revealed that integration of HTP with vacuum has the highest potential in terms of VFA recovery, sludge disintegration, and solid reduction. HTP and vacuum fermentation systems were associated with the highest COD solubilization (45 %), and VFA yield (0.32 g COD/g VSS added). Vacuum fermenters with and without pre-treatment have the highest specific denitrification rates of 7.6 and 7.2 mg NO3-N/g VSS.h, respectively, compared to all other samples and control (acetate). Changes brought about by vacuum fermentation included a shift in the microbial community toward enriching fermenters, mainly Caprothermobacteria and Thermotagea, responsible for VFA production.

Thraustochytrids are heterotrophic marine protists known for their ability to produce valuable lipids such as docosahexaenoic acid (DHA). However, like many non-model organisms, they present challenges for transcriptomic studies due to the limited reliable reference genomes and compatibility with curated protein databases, complicating the respective assembly and annotation. This study presents a de novo transcriptome assembly and functional annotation for the recently isolated thraustochytrid strain Ulkenia visurgensis Lng2 using solely free access software and code available in public domain repositories. The assembled transcriptome presented 45,867 unique gene models, with a total of 66,623 transcripts and a contig N50 of 3162. Functional annotations highlighted high amounts of transcripts related to the biosynthesis of relevant lipidic molecules, as well as stress-adaptive features including catalytic and xenobiotic degrading activity. Likewise, 2381 transcripts were linked to enzymes with potential biotechnological applications. Notably, several transcripts corresponding to uncommon enzymatic activities in thraustochytrids, including laccases, cellulases, and chitinases, were identified. Additionally, evidence for a potential lactamase activity was found, marking the first report of such activity in thraustochytrids. Overall, this study offers a simple free-access procedural strategy for a de novo transcriptome assembly and functional annotation in non-model organisms. These results provide valuable insights into the biotechnological potential of thraustochytrids, while also expanding the limited transcriptomic data available for these protists. Notably, it represents the first transcriptomic analysis of the Ulkenia genus.

Acidification is a key component of digestion throughout metazoans. The gut digestive fluid of many invertebrates is acidified by the vesicular-type H+-ATPase (VHA). In contrast, vertebrates generate acidic gut fluids using the gastric H+/K+-ATPase (HKA), an evolutionary innovation linked with the appearance of a true stomach that greatly improves digestion, absorption and immune function. Hagfishes are the most basal extant vertebrates, and their mechanism of digestive acidification remains unclear. Herein, we report that the stomachless Pacific hagfish (Eptatretus stoutii) acidify their gut using the VHA, and searches of E. stoutii gut transcriptomes and the genome of a closely related hagfish species (E. burgerii) indicate they lack HKA, consistent with its emergence following the 2R whole-genome duplication. Immunostaining revealed prominent VHA presence in the apical membrane of enterocytes and sub-apical expression of both VHA and soluble adenylyl cyclase. Interestingly, akin to vertebrates, VHA was also observed in immature pancreatic-like zymogen granules and was noticeably absent from the mature granules. Furthermore, isolated gut sacs from fed hagfish demonstrate increased VHA-dependent luminal H+ secretion that is stimulated by the cAMP pathway. Overall, these results suggest that the hagfish gut shares the trait of VHA-dependent acidification with invertebrates, while simultaneously performing some roles of the pancreas and intestine of gnathostomes.